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KR20180093396A - Mesenchymal stem cell line useful for developing fibrosis therapeutic agent - Google Patents

Mesenchymal stem cell line useful for developing fibrosis therapeutic agent Download PDF

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KR20180093396A
KR20180093396A KR1020170019416A KR20170019416A KR20180093396A KR 20180093396 A KR20180093396 A KR 20180093396A KR 1020170019416 A KR1020170019416 A KR 1020170019416A KR 20170019416 A KR20170019416 A KR 20170019416A KR 20180093396 A KR20180093396 A KR 20180093396A
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윤병수
김한수
김준환
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Abstract

본 발명은 섬유증 치료제의 개발에 유용한 간엽줄기세포주에 관한 것으로, 구체적으로 TGF-β 또는 PDGF의 처리로 섬유화가 유도되는 것을 특징으로 하는 간성상세포 유래 간엽줄기세포주 ONGHEPA1(KCTC13086BP) 및 이를 이용하여 섬유증 치료제를 스크리닝하는 방법에 관한 것이다.
본 발명의 간엽줄기세포주는 세포 또는 조직의 섬유화 모델로서 뿐만 아니라 새로운 섬유화 치료제의 개발을 위한 세포로서도 매우 유용하게 이용될 수 있으며, 본 발명의 스크리닝 방법에 따르면 다양한 후보물질로부터 정확하고 효율적으로 섬유증 치료제를 스크리닝할 수 있다.The present invention relates to a mesenchymal stem cell line useful for the development of a therapeutic agent for fibrosis. Specifically, the present invention provides a mesenchymal stem cell line ONGHEPA1 (KCTC13086BP), which is characterized in that fibrosis is induced by treatment with TGF-β or PDGF, And a method for screening a therapeutic agent.
The mesenchymal stem cell line of the present invention can be used not only as a fibrosis model of cells or tissues but also as a cell for the development of new fibrosis therapeutic agents. According to the screening method of the present invention, Can be screened.

Description

섬유증 치료제의 개발에 유용한 간엽줄기세포주{Mesenchymal stem cell line useful for developing fibrosis therapeutic agent}The present invention relates to a mesenchymal stem cell line useful for developing a therapeutic agent for fibrosis,

본 발명은 섬유증 치료제의 개발에 유용한 간엽줄기세포주에 관한 것으로, 구체적으로 TGF-β 또는 PDGF의 처리로 섬유화가 유도되는 것을 특징으로 하는 간성상세포 유래 간엽줄기세포주 ONGHEPA1(KCTC13086BP) 및 이를 이용하여 섬유증 치료제를 스크리닝하는 방법에 관한 것이다.The present invention relates to a mesenchymal stem cell line useful for the development of a therapeutic agent for fibrosis. Specifically, the present invention provides a mesenchymal stem cell line ONGHEPA1 (KCTC13086BP), which is characterized in that fibrosis is induced by treatment with TGF-β or PDGF, And a method for screening a therapeutic agent.

섬유증(fibrosis)은 재생이나 발생과정에서 기관이나 조직에 과도한 섬유성 결합조직이 형성되는 질환으로, 이러한 섬유성 결합조직은 정상적인 섬유조직의 형성과는 대조적이다. 기관이나 조직에 섬유성 결합조직이 과도하게 형성되면 조직이 단단해지고 체액의 유입이 감소되는 등 생체 내에서 본래의 기능을 충분히 수행할 수 없게 된다. 원인으로는 부상, 염증, 화상, 방사선, 화학요법, 림프수종 등이 알려져 있다. 이러한 섬유증으로 인한 문제는 섬유성 결합조직이 형성되는 위치에 따라 달라지게 되는데, 주로 간, 분비기관, 폐 등이 손상을 받는다. 섬유증에는 대표적으로 특발성 폐섬유증(idiopathic pulmonary fibrosis, IPF), 골수섬유증(myelo fibrosis), 간섬유증(liver fibrosis) 및 신장섬유증(kidney fibrosis)이 있다.Fibrosis is a disease in which excessive fibrous connective tissue is formed in an organ or tissue during regeneration or development, and this fibrous connective tissue is in contrast to the formation of normal fibrous tissue. If the fibrous connective tissues are excessively formed in the organs or tissues, the tissues become hard and the inflow of the body fluids is reduced, and the in vivo functions can not be sufficiently performed. The causes are injuries, inflammations, burns, radiation, chemotherapy, lymphatic species, and the like. The problem with fibrosis depends on the location of the fibrous connective tissue, mainly the liver, secretory organs, and lungs. Fibroids are typically idiopathic pulmonary fibrosis (IPF), myelo fibrosis, liver fibrosis, and kidney fibrosis.

특발성 폐섬유증은 만성적으로 진행되는 간질성 폐질환의 하나로 병의 경과가 좋지 않고 아직까지 증명된 치료 방법이 없는 질환이다. 폐의 조직검사 결과 벌집모양과 일정하지 않은 모양 등이 확인될 때 진단한다. 서서히 호흡곤란을 유발하며 저산소증 또는 심근경색으로 사망에 이르는 등 경과가 좋지 않다. 현재까지 원인으로 뚜렷하게 입증된 것은 없으나, 환경, 바이러스, 유전, 독성 화합물 등의 다양한 인자로 인해 폐에 염증이 유발되고 이 염증이 치유되는 과정에서 섬유세포가 과도하게 증식하여 폐에 섬유화가 진행되는 것으로 생각되고 있다.Idiopathic pulmonary fibrosis is a chronic progressive interstitial lung disease. It is a disease that has not progressed well and has not yet been proven. Diagnosis is made when histological examination of the lung reveals honeycomb shape and irregular shape. It causes gradual dyspnea and is not good enough to lead to death due to hypoxia or myocardial infarction. Although there has not been any definite evidence to date, it has been shown that various inflammatory factors such as environmental, virus, genetic and toxic compounds lead to inflammation in the lungs. In the process of healing this inflammation, .

골수섬유증은 골수조직의 섬유가 과잉발육되는 질환으로 피를 만드는 기능이 낮아지며 적혈구와 백혈구의 수, 이들의 작용에 변화가 일어나게 된다. 특발성과 속발성으로 구분된다. 이중 특발성은 전신골수의 심각한 섬유증식, 비대 증상을 나타내고, 말초혈중에 유핵적혈구나 유약한 과립구가 나타난다. 원인은 불확실하며 골수의 중독, 염증 등이 원인으로 생각되고 있다. 속발성은 백혈병, 악성림프종, 암골수전이, 화학약품의 중독 등의 경과중에 생긴다. 효과적인 치료제는 아직 개발되지 않은 실정이다.Bone marrow fibrosis is an overgrowth of the bone marrow tissue, which causes a decrease in the ability to make blood and changes in the number of red blood cells and white blood cells and their actions. Idiopathic and secondary. Double idiopathic manifestation of severe fibrosis and hypertrophy of systemic bone marrow, nucleated erythrocytes or glazed granulocytes in peripheral blood. The cause is uncertain, and bone marrow poisoning and inflammation are thought to be the cause. Secondary outbreaks occur during the course of leukemia, malignant lymphoma, cancer marrow metastasis, and chemical poisoning. Effective treatments have not yet been developed.

간섬유증은 간경변증이라고도 한다. 만성적인 염증으로 인해 정상적인 간조직이 재생결절 등의 섬유화 조직으로 바뀌어 간의 기능이 저하되는 질환이다. 만성 B형 간염이나 C형 간염, 과음, 간독성물질 등으로 인해 간의 염증상태가 지속되는 경우에 주로 발생한다. 치료는 증상의 진행을 최대한 늦추는 목표로 이루어지며, 그 원인에 따라 항바이러스제 등이 사용되고 있으나 원인이 다를 경우 그 효과는 미지수이다.Liver fibrosis is also called cirrhosis. It is a disease in which normal liver tissue is replaced with a fibrotic tissue such as regenerative nodule due to chronic inflammation, and liver function is deteriorated. Chronic hepatitis B, hepatitis C, heavy drinking, liver toxicity due to substances such as persistent inflammation of the liver occurs mainly. Treatment is aimed at slowing the progression of symptoms to the utmost, and antiviral drugs are used depending on the cause, but the effect is unknown if the cause is different.

신장섬유증은 세포외 기질이 축적되어 신장에 섬유화가 이루어지는 진행성 질환이며, 사구체 경화증과 세뇨관 간질성 섬유증이 특징이다. 신장의 섬유화로 인해 신장 기능에 부작용이 초래된다. 원인은 외상, 감염, 수술, 환경적인 요인, 화학물질, 방사선 노출 등이 있고, 통증, 배뇨관련문제, 메스꺼움, 구토 등의 증상이 나타날 수 있다. 약물이나 신장 이식으로 증상을 관리하지만 역시 효과적인 치료제의 개발이 필요하다.Renal fibrosis is a progressive disease in which extracellular matrix accumulates and fibrosis occurs in the kidney. It is characterized by glomerulosclerosis and tubular interstitial fibrosis. Fibrosis of the kidneys has side effects on kidney function. Causes include trauma, infection, surgery, environmental factors, chemicals, radiation exposure, pain, urination problems, nausea and vomiting. Management of symptoms by drug or kidney transplantation, however, requires the development of effective treatments.

한편, Grande 등은 최근에 상피간엽이행(Epithelial Mesenchymal Transition, EMT)(이하, 'EMT'라 한다.)의 주요 조절인자인 Twist 혹은 Snail이 신장 섬유화에서 중요한 역할을 담당한다는 연구결과를 제시한 바 있으며, Lovisa 등 또한 EMT가 신장 섬유증에서 중요한 역할을 한다고 제시한 바 있다. EMT는 정상세포가 종양세포로 가면서 중간단계인 세포골격 변화로 인해 세포모양이 이동하기 쉬운 간엽세포(mesenchymal cell)의 형태로 유전적 리프로그래밍(genetic reprogramming)되는 현상을 말한다. 따라서 EMT 관여 단백질의 발현을 억제하면 종양의 전이와 증식을 억제할 수 있다고 생각하여, 다양한 연구자들이 종양 치료제를 개발하기 위하여 이러한 EMT와 관련된 연구를 진행하고 있기도 하다. 이 EMT의 조절인자로는 Twist, Snail, Slug, E-cadherin, vimentin, collagen11 a1 등 약 수백여 개가 알려져 있다.Meanwhile, Grande et al. Have recently presented a study that Twist or Snail, a major regulator of epithelial mesenchymal transition (EMT) (hereinafter referred to as "EMT"), plays an important role in renal fibrosis And Lovisa et al. Have also suggested that EMT plays an important role in renal fibrosis. EMT refers to a phenomenon in which normal cells are genetically reprogrammed in the form of mesenchymal cells, which are likely to migrate due to changes in the cytoskeleton, which is an intermediate stage of tumor cells. Therefore, it is thought that inhibition of the expression of EMT-related proteins can inhibit tumor metastasis and proliferation, and various researchers have been conducting researches related to EMT to develop tumor therapeutics. Some of the regulatory factors for this EMT are Twist, Snail, Slug, E-cadherin, vimentin, and collagen11 a1.

Suzuki 등은 DEC2(BHLHE41)가 이러한 EMT의 조절인자인 Twist의 전사억제인자이고, 이것의 발현을 저해하면 EMT가 활성화되어 악성 종양으로 전환된다는 것을 보고하였다. 또한 Sato 등은 DEC2가 EMT의 조절인자인 Slug의 발현을 억제하고 이에 따라 TGF-β로 유도되는 악성 종양화를 억제한다는 것을 보고하였다. 이밖에도 다양한 연구자들이 DEC2와 같은 EMT의 조절인자가 암전이 등과 관련이 있다는 것을 보고한 바 있다.Suzuki et al. Have reported that DEC2 (BHLHE41) is a transcriptional repressor of Twist, which is a regulator of EMT, and that when it is inhibited, EMT is activated and transformed into a malignant tumor. In addition, Sato et al. Reported that DEC2 inhibited the expression of Slug, a regulator of EMT, and thus inhibited TGF-β-induced malignant tumorigenesis. In addition, various investigators have reported that EMT regulators such as DEC2 are associated with metastasis.

이와 같이 EMT 및 이 EMT의 조절인자에 관한 연구는 대부분 암 또는 종양에 관해서만 진행되어 왔다. 하지만 본 발명자는 기존의 일부 연구결과를 바탕으로 EMT와 섬유증의 연관성에 주목하게 되었고 EMT를 조절할 수 있다면 섬유증을 예방 및 치료할 수도 있을 것으로 기대하였다.Thus, studies on EMT and regulators of this EMT have been conducted mostly for cancer or tumors. However, the present inventor has focused on the relationship between EMT and fibrosis based on some existing research results, and it is expected that fibromatosis can be prevented and treated if EMT can be controlled.

본 발명자는 EMT와 섬유증과의 연관성 및 DEC2와 EMT의 연관성에 주목하고, 유파틸린이 골수세포에서 분화된 대식세포의 DEC2 mRNA를 상향조절한다는 관찰을 통해 유파틸린의 섬유증 치료제로서의 가능성을 연구한 결과, 실제로 유파틸린이 EMT를 억제할 수 있다는 것을 세포모델 및 동물모델을 통해 밝혔고, EMT의 활성화로 기관 또는 조직이 섬유화하는 것을 유파틸린이 효과적으로 억제할 수 있음을 밝힌 바 있다.The present inventors have paid attention to the relationship between EMT and fibrosis and the relationship between DEC2 and EMT and have studied the possibility of the treatment of epothilamine as fibrosis by observing that dietary phytyline upregulates DEC2 mRNA of differentiated macrophages in bone marrow cells In fact, it has been shown through cell models and animal models that EMT can be inhibited by oil tillin, and EMT activation has been shown to effectively inhibit the fibrosis of the organs or tissues.

본 발명자는 이 과정에서 유파틸린과 같은 섬유증 치료제의 섬유증 치료 효과를 보다 정확하고 효율적으로 확인할 수 있는 방법의 필요성을 크게 느끼게 되었으며, 이에 정확하고 효율적인 섬유증 치료제의 스크리닝 방법과 이를 위해 필요한 세포모델을 개발하고자 하였다.In this process, the present inventor has greatly felt the necessity of a method for more accurately and efficiently confirming the therapeutic effect of fibrosis treatment of fibrosis such as epothilone, and accordingly, a screening method of an accurate and efficient fibrosis therapeutic agent and a cell model .

Grande MT, Sanchez-Laorden B, Lopez-Blau C, De Frutos CA, Boutet A, Arevalo M, Rowe RG, Weiss SJ, Lopez-Novoa JM, Nieto MA. Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease. Nat Med. 2015 Sep;21(9):989-97.Grande MT, Sanchez-Laorden B, Lopez-Blau C, De Frutosca, Bouteta, Arevalo M, Rowe RG, Weiss SJ, Lopez-Novoa JM, Nieto MA. Snail1-induced partial epithelial-to-mesenchymal transition drives renal fibrosis in mice and can be targeted to reverse established disease. Nat Med. 2015 Sep; 21 (9): 989-97. Lovisa S, LeBleu VS, Tampe B, Sugimoto H, Vadnagara K, Carstens JL, Wu CC, Hagos Y, Burckhardt BC, Pentcheva-Hoang T, Nischal H, Allison JP, Zeisberg M, Kalluri R. Epithelial-to-mesenchymal transition induces cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med. 2015 Sep;21(9):998-1009.Epidhelial-to-mesenchymal transition. The epithelial-to-mesenchymal transition is defined as the epithelial-to-mesenchymal transition. induced cell cycle arrest and parenchymal damage in renal fibrosis. Nat Med. 2015 Sep; 21 (9): 998-1009. Suzuki M, Sato F, Bhawal UK. The basic helix-loop-helix(bHLH) transcription factor DEC2 negatively regulates Twist1 through an E-box element. Biochem Biophys Res Commun. 2014 Dec 12;455(3-4):390-5.Suzuki M, Sato F, Bhawal UK. The basic helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates Twist1 through an E-box element. Biochem Biophys Res Commun. 2014 Dec 12; 455 (3-4): 390-5. Sato F, Kawamura H, Wu Y, Sato H, Jin D, Bhawal UK, Kawamoto T, Fujimoto K, Noshiro M, Seino H, Morohashi S, Kato Y, Kijima H. The basic helix-loop-helix transcription factor DEC2 inhibits TGF-β-induced tumor progression in human pancreatic cancer BxPC-3 cells. Int J Mol Med. 2012 Sep;30(3):495-501.The basic helix-loop-helix transcription factor DEC2 inhibits the proliferation and proliferation of hepatocytes, including the proliferation of hepatocytes. TGF-β-induced tumor progression in human pancreatic cancer BxPC-3 cells. Int J Mol Med. 2012 Sep; 30 (3): 495-501. Dong Y, Geng Y, Li L, Li X, Yan X, Fang Y, Li X, Dong S, Liu X, Li X, Yang X, Zheng X, Xie T, Liang J, Dai H, Liu X, Yin Z, Noble PW, Jiang D, Ning W. Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice. J Exp Med. 2015 Feb 9;212(2):235-52.Li Y, Li X, Li X, Yan X, Fang Y, Li X, Li X, Li X, Li X, Li X, Zheng X, Xie T, , Noble PW, Jiang D, Ning W. Blocking follistatin-like 1 attenuates bleomycin-induced pulmonary fibrosis in mice. J Exp Med. 2015 Feb 9; 212 (2): 235-52.

따라서 본 발명의 주된 목적은 섬유증 치료제를 정확하고 효율적으로 스크리닝할 수 있는 방법을 제공하는데 있다.Accordingly, a main object of the present invention is to provide a method for accurately and efficiently screening a therapeutic agent for fibrosis.

본 발명의 다른 목적은 상기와 같은 스크리닝 방법에 필요한 세포주를 제공하는데 있다.It is another object of the present invention to provide a cell line necessary for such a screening method.

본 발명의 한 양태에 따르면, 본 발명은 TGF-β(Transforming growth factor beta) 또는 PDGF(Platelet-derived growth factor)의 처리로 섬유화(fibrosis)가 유도되는 것을 특징으로 하는 간성상세포 유래 간엽줄기세포주 ONGHEPA1(KCTC13086BP)을 제공한다.According to one aspect of the present invention, there is provided a hepatic stem cell line derived from hepatic stellate cells, characterized in that fibrosis is induced by treatment of TGF-beta (Transforming growth factor beta) or PDGF (Platelet-derived growth factor) ONGHEPA1 (KCTC13086BP).

본 발명의 다른 양태에 따르면, 본 발명은 상기 ONGHEPA1에 TGF-β 및 PDGF 중에서 선택된 하나의 단백질 및 후보물질을 처리하는 단계; 및 상기 단백질 및 후보물질을 처리한 ONGHEPA1과 후보물질을 처리하지 않은 대조군의 섬유화 정도를 비교하는 단계;를 포함하는 섬유증 치료제의 스크리닝 방법을 제공한다.According to another aspect of the present invention, the present invention relates to a method for treating cancer, comprising the steps of: treating ONGHEPA1 with one protein selected from TGF-? And PDGF and a candidate substance; And comparing the degree of fibrosis of the ONGHEPA1 treated with the protein and the candidate substance and the degree of fibrosis of the control group not treated with the candidate substance.

본 발명의 스크리닝 방법은 특발성 폐섬유증(idiopathic pulmonary fibrosis), 골수섬유증(myelofibrosis), 간섬유증(liver fibrosis) 및 신장섬유증(kidney fibrosis)으로 이루어진 군 중에서 선택된 섬유증에 대한 치료제를 스크리닝하기 위한 방법으로 이용하는 것이 바람직하다.The screening method of the present invention is used as a method for screening a therapeutic agent for fibrosis selected from the group consisting of idiopathic pulmonary fibrosis, myelofibrosis, liver fibrosis and kidney fibrosis .

본 발명의 스크리닝 방법에 있어서, 상기 섬유화 정도는 세포의 형태를 관찰하는 방법을 이용하여 판단하는 것이 바람직하다.In the screening method of the present invention, the degree of fibrosis is preferably determined using a method of observing the morphology of the cells.

본 발명의 스크리닝 방법에 있어서, 상기 단백질 및 후보물질을 처리한 ONGHEPA1과 후보물질을 처리하지 않은 대조군의 유전자의 발현양상을 비교하는 단계;를 더 포함하는 것이 바람직하다.In the screening method of the present invention, it is preferable that the method further comprises the step of comparing the expression patterns of ONGHEPA1 treated with the protein and the candidate substance and the gene of a control group not treated with the candidate substance.

본 발명의 스크리닝 방법에 있어서, 상기 유전자는 Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpinf1, Tlcd2, Frem1, Cald1, Loxl2, Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld, Fam53b, Cav1, Nisch, Fndc1, Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Il18rap, Wbscr17, Col1a1, Synpo, Itgb5, Tnks2, Plod3, Btaf1, Dync1h1, Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Arhgef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, Osbpl9, ERGIC1, Rnf145, Axl, Ltbp2, Ltbp4, Mcfd2 및 Akt1로 이루어진 군 중에서 선택된 하나 또는 둘 이상의 유전자인 것이 바람직하다.In the screening method of the present invention, the gene is selected from the group consisting of Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, , Tlcd2, Frem1, Cald1, Loxl2, Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld , Fam53b, Cav1, Nisch, Fndc1, Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Lig1, Il18rap, Wbscr17, Col1a1, Synpo, , Tnks2, Plod3, Btaf1, Dync1h1, Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Argef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, It is preferably one or two or more genes selected from the group consisting of ERGIC1, Rnf145, Axl, Ltbp2, Ltbp4, Mcfd2 and Aktl.

본 발명의 간엽줄기세포주는 세포 또는 조직의 섬유화 모델로서 뿐만 아니라 새로운 섬유화 치료제의 개발을 위한 세포로서도 매우 유용하게 이용될 수 있으며, 본 발명의 스크리닝 방법에 따르면 다양한 후보물질로부터 정확하고 효율적으로 섬유증 치료제를 스크리닝할 수 있다.The mesenchymal stem cell line of the present invention can be used not only as a fibrosis model of cells or tissues but also as a cell for the development of new fibrosis therapeutic agents. According to the screening method of the present invention, Can be screened.

도 1은 본 발명의 ONGHEPA1의 IHC 분석 결과를 나타낸 것으로, ONGHEPA1이 간성상세포(Hepatic Stellate Cells, HSC)(이하, 'HSC'라 한다.) 계통의 세포라는 것을 알 수 있다. 좌측 : anti-GATA4 항체 반응 양성화를 사용한 IHC 결과, 우측 : anti-CK-18 항체 반응 음성화를 사용한 IHC 결과.
도 2는 본 발명의 ONGHEPA1의 FACS 분석 결과를 나타낸 것으로, ONGHEPA1이 간엽줄기세포로서 생체막 단백질인 CD29, CD44, CD71 및 CD106을 발현한다는 것을 나타낸다.
도 3은 본 발명의 ONGHEPA1에 TGF-β를 처리하여 시간에 따른 세포의 형태를 현미경으로 촬영한 사진이다. None-treated : 별도의 처리없이 ONGHEPA1 세포를 배양한 대조군, TGFβ : TGF-β를 5ng/㎖로 처리한 실험군.
도 4는 본 발명의 ONGHEPA1에 PDGF를 처리하여 시간에 따른 세포의 형태를 현미경으로 촬영한 사진이다. None-treated : 별도의 처리없이 ONGHEPA1 세포를 배양한 대조군, PDGF : PDGF를 5ng/㎖로 처리한 실험군.
도 5는 골수세포에서 분화된 대식세포에서 유파틸린의 DEC2 mRNA 발현 증가 효과를 나타낸 그래프이다.
도 6은 유파틸린의 섬유화 저해 효과를 나타낸 것으로, 각 실험군 중 생쥐 1마리로부터 3부분의 폐조직을 회수 후 masson's trichrome staining하여 현미경으로 촬영한 사진이다. 정상대조군 : 블레오마이신 및 유파틸린을 투여하지 않고 비히클(vehicle) 만을 투여하여 사육한 정상 생쥐, 블레오마이신 투여군 : 블레오마이신을 투여하여 폐섬유화를 유도한 생쥐, 블레오마이신+유파틸린 투여군 : 블레오마이신을 투여하여 폐섬유화를 유도하고 유파틸린(40㎍)을 투여한 생쥐.
도 7은 본 발명의 ONGHEPA1에 섬유화 유도제(TGF-β 또는 PDGF) 및 유파틸린을 처리하고 세포의 형태를 현미경으로 촬영한 사진(각 A 및 B의 상단), 및 면역 형광염색과 DAPI 염색한 결과(각 A 및 B의 하단)이다. A) ONGHEPA1에 별도의 처리를 하지 않거나(Non-treated), TGF-β(5ng/㎖)(TGFβ 24h) 또는 TGF-β+유파틸린(50μM)(TGFβ+eupatilin 24h)을 처리하고 24시간 이후의 세포에 대한 결과, B) ONGHEPA1에 별도의 처리를 하지 않거나(Non-treated), PDGF(5ng/㎖)(PDGF 24h) 또는 PDGF+유파틸린(50μM)(PDGF+eupatilin 24h)을 처리하고 24시간 이후의 세포에 대한 결과.
도 8은 NHLF(normal human lung fibroblast)에 섬유화 유도제(TGF-β 또는 PDGF) 및 유파틸린을 처리하고 세포의 형태를 현미경으로 촬영한 사진(각 A 및 B의 상단), 및 면역 형광염색과 DAPI 염색한 결과(A의 하단)이다. A) NHLF에 별도의 처리를 하지 않거나(Non-treated), TGF-β(5ng/㎖)(TGFβ 48h) 또는 TGF-β+유파틸린(50μM)(TGFβ+eupatilin 48h)을 처리하고 48시간 이후의 세포에 대한 결과, B) ONGHEPA1에 별도의 처리를 하지 않거나(Non-treated), PDGF(5ng/㎖)(PDGF 48h) 또는 PDGF+유파틸린(50μM)(PDGF+eupatilin 48h)을 처리하고 48시간 이후의 세포에 대한 결과.
도 9는 본 발명의 ONGHEPA1에 별도의 처리를 하지 않거나(DMEM), 섬유화 유도제(TGF-β 또는 PDGF)(5ng/㎖)만을 처리하거나(TGFβ 24h, PDGF 24h), 섬유화 유도제 및 유파틸린(50μM)을 함께 처리하거나(TGFβ+eupatilin 24h, PDGF+eupatilin 24h), 섬유화 유도제 및 ONGE200(50μM)을 함께 처리하고(TGFβ+ONGE200 24h, PDGF+ONGE200 24h) 24시간 이후에 세포의 형태를 현미경으로 촬영한 사진이다.
도 10은 본 발명의 ONGHEPA1에 별도의 처리를 하지 않거나(DMEM), 섬유화 유도제(TGF-β)(5ng/㎖)만을 처리하고(TGFβ 24h) 24시간 이후에 세포의 형태를 현미경으로 촬영한 사진 및 섬유화 유도제를 처리한 다음 24시간 이후 유파틸린(50μM) 또는 ONGE200(50μM)을 처리하고(TGFβ 24h+eupatilin 24h, TGFβ 24h+ONGE200 24h) 24시간 이후에 세포의 형태를 현미경으로 촬영한 사진이다.
도 11은 본 발명의 ONGHEPA1에 섬유화 유도제(TGF-β 또는 PDGF)(5ng/㎖)만을 처리하거나(TGF-b, PDGF), 섬유화 유도제 및 유파틸린(50μM)을 함께 처리하거나(Eup), 섬유화 유도제 및 ONGE200(50μM)을 함께 처리하고(ONGE200) total RNA를 정제하여 real-time PCR을 통해 각 EMT 관련 유전자(Col11A1, slit3, Axl, Postn, Fn1 또는 Aurka)의 발현 양상을 분석한 그래프이다. A) 섬유화 유도제로 TGF-β를 이용한 실험의 결과, B) 섬유화 유도제로 PDGF를 이용한 실험의 결과.
도 12는 NHLF에 별도의 처리를 하지 않거나(Non treated control), PDGF(5ng/㎖) 만을 처리하거나(PDGF_48h, PDGF_72h), PDGF 및 유파틸린(50μM)을 함께 처리하고(Eup_48h, Eup_72h) total RNA를 정제하여 real-time PCR을 통해 각 EMT 관련 유전자(Col11A1, vimentin 또는 periostin)의 발현 양상을 분석한 그래프이다.
도 13은 대조군 ONGHEPA1, 섬유화를 유도한 ONGHEPA1 및 섬유화를 유도하고 유파틸린을 처리한 ONGHEPA1의 transcriptome을 분석한 결과를 나타낸 것이다. 대조군 : ONGHEPA1, TGFβ : 배양액에 TGF-β를 첨가하고 24시간 동안 ONGHEPA1을 배양한 대조군, TGFβ+Eup : 배양액에 TGF-β 및 유파틸린을 첨가하고 24시간 동안 ONGHEPA1을 배양한 실험군.
도 14는 정상적인 ONGHEPA1, 섬유화를 유도한 ONGHEPA1 및 섬유화를 유도하고 유파틸린을 처리한 ONGHEPA1에서 발현이 변화되는 유전자의 유전자 induction fold와 p-value를 비교하여 나타낸 volcano plot이다.
도 15는 정상적인 ONGHEPA1, 섬유화를 유도한 ONGHEPA1 및 섬유화를 유도하고 유파틸린을 처리한 ONGHEPA1에서 발현이 유의적으로 변화되는 유전자들의 interactome을 unbiased manner로 분석하여 나타낸 것이다.FIG. 1 shows the results of IHC analysis of ONGHEPA1 of the present invention. It can be seen that ONGHEPA1 is a cell line of hepatic stellate cells (HSC) (hereinafter, referred to as HSC). Left: IHC results using anti-GATA4 antibody positive, right: IHC results using anti-CK-18 antibody response negative.
Fig. 2 shows the results of FACS analysis of ONGHEPA1 of the present invention. It shows that ONGHEPA1 expresses biomembrane proteins CD29, CD44, CD71 and CD106 as mesenchymal stem cells.
Fig. 3 is a photograph of ONGHEPA1 of the present invention, which is obtained by treating TGF-beta with time and observing the morphology of the cell with time. Fig. None-treated: Control group treated with 5ng / ml of TGFβ: TGF-β as a control group in which ONGHEPA1 cells were cultured without any treatment.
FIG. 4 is a photograph of the ONGHEPA1 of the present invention taken by PDGF and photographed with a microscope. None-treated: a control group in which ONGHEPA1 cells were cultured without any treatment, and an experimental group treated with PDGF: PDGF at 5 ng / ml.
FIG. 5 is a graph showing the effect of increasing the DEC2 mRNA expression of the phylactin in macrophages differentiated from bone marrow cells.
FIG. 6 shows the effect of the inhibition of fibrotylation of milk tilline. In the experiment group, three parts of lung tissue were collected from one mouse and masson's trichrome staining was taken with a microscope. Normal control group: normal mice fed with vehicle alone without administration of bleomycin and yupatiline, bleomycin treated group: mice induced to pulmonary fibrosis by administration of bleomycin, bleomycin plus yupatiline treated group: bleomycin Mice were induced with pulmonary fibrosis and treated with 40 mg of efavirenz.
FIG. 7 shows photographs (tops of A and B) of ONGHEPA1 of the present invention treated with a fibrosis inducer (TGF-? Or PDGF) and? -Fatilin, and the morphology of cells was photographed (Bottom of each of A and B). A) TGF-β (5 ng / ㎖) (TGFβ 24h) or TGF-β + ufatilin (50 μM) (TGFβ + eupatilin 24h) was treated with ONGHEPA1 without treatment (PDGF + eupatilin 24h) or PDGF (5 ng / ㎖) (PDGF 24h) or PDGF + Results for subsequent cells.
FIG. 8 shows photographs (tops of A and B) obtained by treating a normal human lung fibroblast (NHLF) with a fibrogen inducer (TGF-β or PDGF) The result of dyeing (bottom of A). A) NHLF treated or non-treated with TGF-β (5 ng / ㎖) (TGFβ 48h) or TGF-β plus taphine (50 μM) (TGFβ + eupatilin 48h) (PDGF + eupatilin 48h) was treated with PDGF (5 ng / ㎖) (PDGF 48h) or PDGF + yupatilin (48 μM) and treated with ONGHEPA1 for 48 hours Results for subsequent cells.
FIG. 9 is a graph showing the effect of the present invention on ONGHEPA1 alone (DMEM), fibrosis inducing agent (TGF-? Or PDGF) (5ng / ml) alone (TGF? 24h, PDGF 24h), fibrosis inducer and 50? ) Were treated together (TGFβ + eupatilin 24h, PDGF + eupatilin 24h), fibrosis inducer and ONGE200 (50 μM) together (TGFβ + ONGE200 24h, PDGF + ONGE200 24h) It's a picture.
FIG. 10 is a photograph of the ONGHEPA1 of the present invention taken after 24 hours after treatment with only DMEM, fibrosis inducing agent (TGF-β) (5 ng / (TGFβ 24h + eupatilin 24h, TGFβ 24h + ONGE200 24h) after 24 hours of treating the cells with a culture supernatant, .
Fig. 11 is a graph showing the results of treatment of ONGHEPA1 of the present invention with fibrogenesis inducer (TGF-β or PDGF) (5 ng / ml) alone (TGF-b, PDGF), fibrosis inducer and oil tiline (Col11A1, Slit3, Axl, Postn, Fn1 or Aurka) through real-time PCR by treating the total RNA (ONGE200) together with the inducer and ONGE200 (50 μM) together. A) TGF-β as a fibrosis inducer, and B) PDGF as a fibrosis inducer.
Fig. 12 shows the results of treatment of NHLF (Non treated control), treatment of PDGF (5 ng / ml) alone (PDGF_48h, PDGF_72h), treatment of PDGF and yaffatilin (50 μM) (Eup_48h, Eup_72h) (Col11A1, vimentin, or periostin) by real-time PCR.
Figure 13 shows the results of analysis of the control ONGHEPA1, fibrosis-induced ONGHEPA1, and transcriptome of ONGHEPA1 induced fibrosis and treated with efavatillin. Control group: ONGHEPA1, TGFβ: a control group in which ONGHEPA1 was cultured for 24 hours with addition of TGF-β to the culture medium. TGFβ + Eup: TGF-β and Ephatilin were added to the culture medium and ONGHEPA1 was cultured for 24 hours.
Fig. 14 is a volcano plot showing normal ONGHEPA1, ONGHEPA1 inducing fibrosis, and gene induction fold and p-value of a gene whose expression is changed in ONGHEPA1, which induces fibrosis and treated with efavatiline.
Fig. 15 shows the normal ONGHEPA1, ONGHEPA1 inducing fibrosis, and the interactome of the genes whose expression is significantly changed in inducible fibrosis and ONGHEPA1 treated with efavatilline in an unbiased manner.

본 발명의 간엽줄기세포주 ONGHEPA1은 한국생명공학연구원의 생물자원센터(KCTC)에 수탁번호 KCTC13086BP로 기탁되어 있다.The mesenchymal stem cell line ONGHEPA1 of the present invention is deposited under the accession number KCTC13086BP in the KCTC of the Korea Research Institute of Bioscience and Biotechnology.

본 발명의 ONGHEPA1은 마우스의 간성상세포(Hepatic Stellate Cells, HSC) 류의 간엽줄기세포(mesenchymal stem cell, MSC)(이하, 'MSC'라 한다.)이며, 무한 증식할 수 있는 세포이다.The ONGHEPA1 of the present invention is a mesenchymal stem cell (MSC) (hereinafter referred to as "MSC") of Hepatic Stellate Cells (HSC) of a mouse and is an endlessly proliferating cell.

TGF-β(Transforming growth factor beta) 또는 PDGF(Platelet-derived growth factor)를 처리하는 간단한 방법으로 섬유화가 유도될 수 있어 세포 섬유화 모델로서 뿐만 아니라, 섬유화 치료증 스크리닝을 위한 세포로서도 매우 유용하다. 특히 ONGHEPA1은 간에서 유래된 세포이기 때문에 간섬유증의 모델로 이용할 수 있는데, 아직까지 마땅한 간섬유증 모델이 없기 때문에 그 가치는 매우 높다고 판단된다.Fibrosis can be induced by a simple method of treating TGF-beta (Transforming growth factor beta) or PDGF (Platelet-derived growth factor), which is very useful not only as a cell fibrosis model but also as a cell for fibrosis treatment screening. Since ONGHEPA1 is a liver-derived cell, it can be used as a model for hepatic fibrosis.

본 발명의 ONGHEPA1은 DMEM 배지에서 배양할 수 있으나 이에 제한되는 것은 아니다. FBS(fetal bovine serum)가 포함되거나 포함되지 않은 상태에서 배양할 수 있으며, 오염방지를 위해 penicillin, streptomycin과 같은 항생제를 포함시켜 배양하는 것이 바람직하다. 배양온도는 37℃ 정도로 하는 것이 바람직하며, 5% 정도의 CO2 상태에서 배양하는 것이 좋다.The ONGHEPA1 of the present invention can be cultured in a DMEM medium, but is not limited thereto. It can be cultured with or without FBS (fetal bovine serum), and it is preferable to cultivate it with antibiotics such as penicillin and streptomycin in order to prevent contamination. The incubation temperature is preferably about 37 캜, and it is preferable to culture in a CO 2 state of about 5%.

본 발명은 상기와 같은 ONGHEPA1을 이용한 섬유증 치료제 스크리닝 방법을 제공하는데, 이 스크리닝 방법은 ONGHEPA1에 TGF-β 및 PDGF 중에서 선택된 하나의 단백질 및 후보물질을 처리하는 단계; 및 상기 단백질 및 후보물질을 처리한 ONGHEPA1과 후보물질을 처리하지 않은 대조군의 섬유화 정도를 비교하는 단계;를 포함하는 것을 특징으로 한다.The present invention provides a method for screening a therapeutic agent for fibrosis using ONGHEPA1 as described above, which comprises treating ONGHEPA1 with one protein selected from TGF-? And PDGF and a candidate substance; And comparing the degree of fibrosis between the ONGHEPA1 treated with the protein and the candidate substance and the control group not treated with the candidate substance.

상기 TGF-β, PDGF 또는 후보물질을 처리하는 방법으로는 ONGHEPA1의 배양액에 후보물질을 현탁하거나 완충액에 후보물질을 현탁하여 ONGHEPA1에 접촉시키는 방법 등을 이용할 수 있다. 각각의 농도는 경우에 따라 적절히 선택하여 적용할 수 있으나, 본 발명에 따르면 섬유화를 유도하기 위한 TGF-β 또는 PDGF는 2 ~ 10ng/㎖가 바람직하며, 보다 바람직하게는 5ng/㎖의 농도가 좋다.As a method of treating the TGF-beta, PDGF or candidate substance, a method of suspending a candidate substance in ONGHEPA1 culture medium or suspending a candidate substance in a buffer solution and contacting the same with ONGHEPA1 can be used. The concentration of TGF-beta or PDGF for inducing fibrosis is preferably 2 to 10 ng / ml, more preferably 5 ng / ml, in order to induce fibrosis according to the present invention .

섬유화 유도제인 TGF-β 또는 PDGF와 후보물질의 처리는 동시에 또는 서로 시점을 달리하여 이루어질 수 있다. 예를 들어, 섬유화 유도제와 후보물질을 동시에 처리하는 방법, 섬유화 유도제를 먼저 처리한 다음 일정 시간 이후에 후보물질을 처리하는 방법, 후보물질을 먼저 처리한 다음 일정 시간 이후에 섬유화 유도제를 처리하는 방법을 이용할 수 있다. 섬유화 유도제를 먼저 처리하는 경우에는 섬유화가 어느 정도 진행된 이후에 후보물질의 작용이 이루어지는 것이므로 치료효과를 보다 명확하게 조사할 수 있으며, 후보물질을 먼저 처리하는 경우에는 예방효과를 보다 명확하게 조사할 수 있다.Treatment of the candidate agent with the fibrosis inducing agent TGF-beta or PDGF may be performed simultaneously or at different time points. For example, a method of simultaneously treating a fibrosis inducing agent and a candidate substance, a method of treating a fibrosis inducing agent first and then a candidate substance after a certain period of time, a method of treating a candidate agent first, Can be used. When the fibrogenesis inducer is first treated, since the action of the candidate substance is performed after the fibrogenesis has progressed to some extent, the therapeutic effect can be more clearly investigated. In case of treating the candidate substance first, have.

본 발명의 스크리닝 방법에 적용할 수 있는 후보물질에는 별도의 제한이 없다. 즉, 천연물질과 인공적인 합성화합물 모두를 적용할 수 있으며, 정제된 화합물이나 추출물의 형태 등에도 제한없이 적용할 수 있을 것으로 판단된다.There is no particular limitation on the candidate substance applicable to the screening method of the present invention. In other words, both natural materials and artificial synthetic compounds can be applied, and it is considered that they can be applied without limitation to the form of purified compounds or extracts.

본 발명의 스크리닝 방법에서는 TGF-β 및 PDGF 중에서 선택된 하나의 단백질, 즉 섬유화 유도제 및 후보물질을 처리한 ONGHEPA1과 후보물질을 처리하지 않은 대조군의 섬유화 정도를 비교하는데, 이때 섬유화 정도는 세포의 형태를 관찰하는 방법을 이용하여 판단할 수 있다. 세포의 형태는 현미경을 통해 관찰할 수 있다. 섬유화가 유도되면 ONGHEPA1은 근섬유아세포(myofibroblast)로 분화되고 세포외 기질 단백질(extracellular matrix protein, ECM)을 세포 사이에 분비하여 세포의 움직임이 둔화되며 αSMactinin(alpha smooth muscle actinin)이 과발현되어 세포 골격이 경화된다. 따라서 세포의 형태가 섬유화 유도제를 처리하지 않은 것에 비해 선명하고 길쭉한 형태를 나타낸다. 이러한 세포의 형태를 비교함으로써 섬유화 정도를 판단할 수 있다. 후보물질을 처리하지 않은 대조군에 비해 섬유화 정도가 덜하거나 섬유화 유도제와 후보물질 모두를 처리하지 않은 ONGHEPA1의 세포형태에 근접하다면 이때의 후보물질이 섬유화 치료 효과가 있다고 볼 수 있다. 섬유증 치료 효과가 우수한 유파틸린을 양성대조군으로 사용하여 후보물질과 비교한다면 섬유증 치료제를 보다 정확하게 스크리닝할 수 있으며, 후보물질의 활성정도도 결정할 수 있다.In the screening method of the present invention, ONGHEPA1 treated with one protein selected from TGF-β and PDGF, that is, a fibrogen inducer and a candidate substance, and a control group not treated with a candidate substance are compared to each other in degree of fibrosis, It can be judged by using an observation method. The morphology of the cells can be observed through a microscope. When fibrosis is induced, ONGHEPA1 is differentiated into myofibroblasts and secretes extracellular matrix protein (ECM) between cells, resulting in slowed cell movement and overexpressing αSMactinin (alpha smooth muscle actinin) Cured. Therefore, the morphology of the cells shows a sharp and elongated morphology compared to the fibrosis inducing agent. The degree of fibrosis can be determined by comparing the morphology of these cells. If the degree of fibrosis is lower than that of the control group without the candidate substance, or the cell type of ONGHEPA1 which is not treated with both the fibrosis inducing agent and the candidate substance is close to the cell type, then the candidate substance may be effective for treating fibrosis. When compared with the candidate substance using the tiline which is excellent in the treatment effect of fibrosis as a positive control, the fibrosis treatment agent can be screened more accurately and the degree of activity of the candidate substance can be determined.

상기와 같이 섬유화 정도를 비교하는 것에 추가로 유전자의 발현양상을 비교하는 방법을 이용하면 보다 정확하고 효과적으로 섬유증 치료제를 스크리닝할 수 있다. 유전자의 발현양상은 real-time PCR 등의 방법을 이용하여 확인할 수 있으나 이에 제한되는 것은 아니다. 본 발명에 따르면 섬유화가 유도된 ONGHEPA1에서 섬유증 치료 효과가 우수한 유파틸린에 의해 여러 유전자의 발현이 조절되며, 이러한 유전자들 중에는 EMT(Epithelial Mesenchymal Transition)에 연관된 유전자가 있는 것으로 나타났다. 유파틸린을 이용한 실험을 통해 EMT가 섬유화에 매우 중요한 역할을 한다는 것을 증명하였으므로, 후보물질의 처리에 따른 이러한 EMT 관련 유전자의 발현양상을 비교한다면 보다 정확하고 효과적으로 섬유증 치료제를 스크리닝할 수 있다. 이러한 EMT 관련 유전자들 중에서 특히 Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpinf1, Tlcd2, Frem1, Cald1, Loxl2, Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld, Fam53b, Cav1, Nisch, Fndc1, Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Il18rap, Wbscr17, Col1a1, Synpo, Itgb5, Tnks2, Plod3, Btaf1, Dync1h1, Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Arhgef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, Osbpl9, ERGIC1, Rnf145, Axl, Ltbp2, Ltbp4, Mcfd2 및 Akt1로 이루어진 군 중에서 선택된 하나 또는 둘 이상의 유전자의 발현양상을 비교하는 것이 바람직하다. 이들 유전자는 ONGHEPA1에서 섬유화가 유도되면 발현이 상향조절되고 유파틸린이 처리되면 발현이 매우 현저하게 하향조절되는 유전자들이다.By comparing the degree of expression of the gene in addition to the degree of fibrosis as described above, the therapeutic agent for fibrosis can be more accurately and effectively screened. The expression pattern of the gene can be confirmed by a method such as real-time PCR, but is not limited thereto. According to the present invention, expression of several genes is regulated by fibrotic induction-mediated ONGHEPA1, which is excellent in the treatment of fibrosis, and there is a gene related to EMT (Epithelial Mesenchymal Transition) among these genes. Since EMT has been shown to play a very important role in fibrosis through experiments with the use of tiline, comparing the expression patterns of these EMT-related genes according to the treatment of candidate substances can provide a more accurate and effective screening of fibrosis therapeutic agents. Among these EMT-related genes, Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpinf1, Tlcd2, Frem1 , Cald1, Loxl2, Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld, Fam53b, Cav1 , Nisch, Fndc1, Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Il18rap, Wbscr17, Col1a1, Synpo, Itgb5, , Btaf1, Dync1h1, Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Argef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, Osbpl9, ERGIC1, Rnf145, It is preferable to compare the expression patterns of one or more genes selected from the group consisting of Axl, Ltbp2, Ltbp4, Mcfd2 and Aktl. These genes are genes whose expression is upregulated when fibrosis is induced in ONGHEPA1 and whose expression is markedly downregulated when treated with efavetillin.

본 발명의 스크리닝 방법에 따르면 특발성 폐섬유증(idiopathic pulmonary fibrosis), 골수섬유증(myelofibrosis), 간섬유증(liver fibrosis) 및 신장섬유증(kidney fibrosis)으로 이루어진 군 중에서 선택된 섬유증에 대한 치료제를 스크리닝할 수 있다. 비록 ONGHEPA1이 간에서 유래된 세포이기는 하지만, 섬유화가 유도될 때 발현이 상향조절되고 섬유화가 억제될 때 발현이 매우 현저하게 하향조절되는 유전자들 중 대부분이 EMT에 관여하는 유전자들이며, 유파틸린을 이용한 실험을 통해 이 EMT가 다른 조직이나 세포의 섬유화에도 관여한다는 것을 증명하였으므로, 본 발명의 스크리닝 방법에 따르면 간섬유증 이외에 상기와 같은 다른 섬유증에 대한 치료제 또한 정확하고 효과적으로 스크리닝할 수 있다.According to the screening method of the present invention, a therapeutic agent for fibrosis selected from the group consisting of idiopathic pulmonary fibrosis, myelofibrosis, liver fibrosis, and kidney fibrosis can be screened. Although ONGHEPA1 is a liver-derived cell, most of the genes whose expression is down-regulated very significantly when expression is upregulated and fibrosis is inhibited when fibrosis is induced are genes that are involved in EMT, Experiments have shown that this EMT also participates in the fibrosis of other tissues or cells. Therefore, the screening method of the present invention can accurately and effectively screen therapeutic agents for other fibrosis as well as hepatic fibrosis.

이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. These embodiments are only for illustrating the present invention, and thus the scope of the present invention is not construed as being limited by these embodiments.

실시예 1. ONGHEPA1 제작Example 1. Production of ONGHEPA1

C57BL/6 마우스의 간조직을 떼어낸 후 단세포 현탁액(single cell suspension)을 만들어 계속적인 배양을 통해 무한 증식하는 세포를 얻고, RT-PCR(reverse transcription polymerase chain reaction), IHC(immunohistochemistry) 및 FACS(fluorescence-activated cell sorting) 실험을 통해 HSC 류의 MSC 세포주를 선별하였다.After the liver tissue of C57BL / 6 mice was detached, a single cell suspension was prepared and cells were subjected to continuous culture to obtain endogenous proliferation cells. The cells were subjected to reverse transcription polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) HSC-like MSC cell lines were screened by fluorescence-activated cell sorting.

HSC는 일반적인 간세포(hepatocytes)와는 다르므로 RT-PCR을 통해 알부민 음성인 세포를 선별하고, anti-GATA4와 anti-CK-18 항체를 이용한 IHC 분석을 통해 GATA4 및 CK-18, 즉 endo/ectodermal 마커가 발현되는 세포를 선별함으로써 HSC를 선별하고자 하였다.Since HSC is different from normal hepatocytes, albumin-negative cells were selected by RT-PCR and IHC analysis using anti-GATA4 and anti-CK-18 antibodies revealed GATA4 and CK-18, that is, endo / ectodermal markers HSCs were selected by screening cells expressing HSCs.

또한 MSC 마커, 즉 CD29, CD44, CD71 및 CD106에 대한 항체를 이용한 FACS 분석을 통해 상기 MSC 마커가 세포의 표면에 발현되는 세포를 선별함으로써 MSC를 선별하고자 하였다.The MSC markers were selected by screening FACS analysis using antibodies against MSC markers, namely CD29, CD44, CD71 and CD106.

이의 결과, 상기의 모든 조건을 만족하는 세포주를 선별할 수 있었으며(도 1 및 2 참조), 이 세포주를 ONGHEPA1이라고 명명하고 한국생명공학연구원의 생물자원센터(KCTC)에 기탁하여 수탁번호(KCTC13086BP)를 부여받았다.As a result, a cell line satisfying all the above conditions was selected (refer to FIGS. 1 and 2), and this cell line was named ONGHEPA1 and deposited with KCTC of the Korea Biotechnology Research Institute (KCTC) to obtain the accession number (KCTC13086BP) .

실시예 2. ONGHEPA1의 섬유화 특성 분석Example 2. Analysis of fibrosis characteristics of ONGHEPA1

간섬유화의 가장 중요한 원인 세포는 HSC이다. HSC는 여러 세포로 분화되며 그 중 하나가 근섬유아세포(myofibroblast)이다. 또한 HSC는 다량의 세포외 기질 단백질(extracellular matrix protein, ECM)을 세포 사이에 분비하여 세포의 움직임을 둔화시킨다. Collagen α1이 가장 많은 ECM 단백질이다. 그리고 동시에 섬유아세포에 αSMactinin이 과발현되어 세포 골격을 경화시킨다.The most important cause of liver fibrosis is HSC. HSCs are differentiated into multiple cells, one of which is myofibroblast. In addition, HSC secretes a large amount of extracellular matrix protein (ECM) between cells, which slows cell movement. Collagen α1 is the most abundant ECM protein. At the same time, αSMactinin is overexpressed in fibroblasts to cure the cytoskeleton.

상기와 같은 HSC의 특성을 이용하여 ONGHEPA1의 섬유화 특성을 확인하고자 하였다. 섬유화를 유도하기 위해 TGF-β(5ng/㎖) 또는 PDGF(5ng/㎖)를 처리하고 경과된 시간에 따라 세포의 형태를 관찰한 결과, TGF-β 또는 PDGF 처리 후 6 ~ 8시간이 경과된 시점부터 섬유화 조짐이 나타났고, 24시간이 경과된 시점에서 상당한 섬유화가 진행되었으며 48시간이 경과된 시점에서는 더욱 섬유화가 진행되었음을 확인하였다(도 3 및 4 참조).The characteristics of ONGHEPA1 fibrosis were examined using the characteristics of HSC as described above. The cells were treated with TGF-β (5 ng / ㎖) or PDGF (5 ng / ㎖) to induce fibrosis and the morphology of the cells was observed at 6 to 8 hours after TGF-β or PDGF treatment Fibrosis was observed from the time point, and significant fibrosis progressed at the lapse of 24 hours, and fibrosis progressed further at 48 hours (see FIGS. 3 and 4).

실시예 3. 섬유증 치료제 스크리닝에서 ONGHEPA1의 이용 가능성 검증Example 3. Availability of ONGHEPA1 in screening for fibrosis treatment

3-1. ONGHEPA1의 이용 가능성 검증에 사용한 유파틸린의 섬유증 치료 효과3-1. Effectiveness of the treatment of fibrotic syndrome in the use of ONGHEPA1

섬유증 치료제의 스크리닝에서 ONGHEPA1의 이용 가능성을 검증하기 위해 섬유증 치료제로 사용한 유파틸린의 섬유증 치료 효과는 다음 실시예 3-1-1 내지 3-1-2와 같은 실험을 통해 증명되었다. 유파틸린의 화학구조는 다음 화학식 1과 같다.In order to verify the availability of ONGHEPA1 in the screening of fibrosis therapeutic agents, the effect of treatment with the fibrotic effect of the use of the medicament for the treatment of fibrosis was demonstrated through the same experiments as the following Examples 3-1-1 to 3-1-2. The chemical structure of the < RTI ID = 0.0 >

[화학식 1][Chemical Formula 1]

Figure pat00001
Figure pat00001

3-1-1. 유파틸린의 DEC2 발현 증가 효과3-1-1. DEC2 expression increase effect

DEC2는 EMT의 조절인자인 Twist 및 Slug의 전사억제인자로 알려져 있다. 따라서 DEC2의 발현이 증가하면 Twist 및 Slug 등 EMT 조절인자의 전사가 억제되고, 이에 따라 EMT가 억제되기 때문에 조직이 섬유화되는 것이 억제될 수 있다. 이에 유파틸린에 의해 DEC2의 발현이 증가하는지를 쥐 골수세포(mouse bone marrow cells, MBMC)를 이용하여 확인하였다.DEC2 is known as a transcriptional repressor of Twist and Slug, the regulators of EMT. Therefore, when the expression of DEC2 is increased, transcription of EMT regulatory factors such as twist and slug is inhibited, and EMT is suppressed, so that fibrosis of tissue can be suppressed. The expression of DEC2 by the tiline was confirmed by mouse bone marrow cells (MBMC).

MBMC에 M-CSF(macrophage-colony stimulating factor)와 RANK(receptor activator of NFκB) 리간드(RANKL)를 처리하여 활성화시키고, 여기에 유파틸린을 50μM의 농도로 처리하여 4일간 배양한 후 DEC2의 발현양상을 확인하였다.MBMC was treated with M-CSF (macrophage-colony stimulating factor) and RANK (receptor activator of NFκB) ligand (RANKL) to induce activation of DEC2 after culturing for 4 days at a concentration of 50 μM Respectively.

이의 결과, 도 5에서와 같이 유파틸린은 DEC2의 mRNA 발현을 7 ~ 8배 증가시키는 DEC2 유도제(inducer)로 작용하는 것으로 나타났다.As a result, as shown in FIG. 5, it was shown that the effect of DEC2 inducer was enhanced by 7 to 8-fold increase in mRNA expression of DEC2.

3-1-2. 블레오마이신으로 유도된 폐조직의 섬유화에서 유파틸린의 효과3-1-2. The Effect of Phosphatiline on the Fibromycin-induced Pulmonary Tissue Fibrosis

실제 동물모델을 사용하여 유파틸린이 실제로 조직 섬유화를 억제할 수 있는지를 확인하였다. 5주령의 수컷 C57BL/6J 생쥐(체중 18.2 ~ 20.5g)(KOATECH, 한국)를 실험동물로 사용하였으며, 실험동물은 각 군당 5마리씩으로 다음 표 1과 같은 군으로 나누었다.Using real animal models, we confirmed that the efavirenz could actually inhibit tissue fibrosis. Five-week-old male C57BL / 6J mice (body weight 18.2-20.5g) (KOATECH, Korea) were used as experimental animals, and the experimental animals were divided into five groups as shown in Table 1 below.

실험군Experimental group 블레오마이신 투여Bleomycin administration 유파틸린 투여Yupatiline administration 정상대조군Normal control group -- -- 블레오마이신 투여군Bleomycin treated group 40㎍/head40 μg / head vehiclevehicle 블레오마이신+유파틸린 1㎍ 투여군1 μg of bleomycin plus yupatiline 40㎍/head40 μg / head 1㎍/20㎕1 / / 20 ㎕ 블레오마이신+유파틸린 5㎍ 투여군5 μg of bleomycin plus yupatiline 40㎍/head40 μg / head 5㎍/20㎕5 / / 20 ㎕ 블레오마이신+유파틸린 10㎍ 투여군10 μg of bleomycin plus yupatiline 40㎍/head40 μg / head 10㎍/20㎕10 / / 20 ㎕ 블레오마이신+유파틸린 20㎍ 투여군20 μg of bleomycin plus yupatiline 40㎍/head40 μg / head 20㎍/20㎕20 / / 20 ㎕ 블레오마이신+유파틸린 40㎍ 투여군Bleomycin plus 40 ug of yupatiline 40㎍/head40 μg / head 40㎍/20㎕40 / / 20 ㎕

실험동물은 SPF(Specific Pathogen Free), BSL(Bio Safety Level) 2등급시설에서 폴리술폰(polysulfone) 재질, 369L x 156W x 132H(mm)(EU, USA, UK GL compliance) 규격의 사육상자를 사용하여 사육하였다. 사육상자 당 수용동물 수는 검역·순화기간에 2 ~ 3마리, 시험기간에 2 ~ 3마리로 하였고, 온도 22 ± 2℃, 상대습도 50.0 ± 15.0%, 환기횟수 10 ~ 20회/시간, 명암주기(조명시간) 12시간/일(07:00 ~ 19:00), 조도 150 ~ 300Lux의 조건 하에서 사육하였다.Experimental animals used polysulfone material, 369L x 156W x 132H (mm) (EU, USA, UK GL compliance) breeding box in SPF (Specific Pathogen Free) and BSL (Bio Safety Level) Respectively. The number of animals per cage was 2 ~ 3 in the quarantine period, 2 ~ 3 in the test period, temperature 22 ± 2 ℃, relative humidity 50.0 ± 15.0%, number of ventilation 10 ~ 20 times / (Illumination time) 12 hours / day (07:00 ~ 19:00), and illumination of 150 ~ 300 Lux.

폐섬유화를 유도하기 위해 Kremer 등, Laxer 등 및 Berkman 등의 기관내 주입(intratracheal instillation, IT) 방법에 따라 블레오마이신(bleomycin) 용액을 기관(trachea)을 통하여 직접 폐 속에 주입하는 방법을 사용하였다. 즉, C57BL/6J 생쥐를 70% N2O와 30% O2 가스 및 1.5% isoflurane으로 흡입마취한 상태에서 전경부의 피부를 절개하고 근육을 정리하여 기관을 노출시킨 다음 안과용 수술가위로 기관을 조금 절개하였다. 앞을 둥글게 만든 19 gauge 주사바늘을 장착한 1㎖ 주사기를 사용하여 블레오마이신을 녹인 증류수용액 50㎕를 절개된 기관을 통하여 직접 폐 속으로 단번에 주입하였다. 주입 직후 곧바로 절개한 전경부의 피부를 봉합하고 마취에서 깨어나게 한 다음 일반사육 케이지에 담아 사육하였다. 블레오마이신의 투여는 비디오 점적기(visual instillobot)를 이용하여 수행하였으며, 블레오마이신-HCl 40㎍/50㎕을 1회 투여하여 12일간의 폐섬유화 질환 유도기간을 설정하였다.To induce pulmonary fibrosis, a bleomycin solution was directly injected into the lungs through a trachea according to the intratracheal instillation (IT) method of Kremer et al., Laxer et al. And Berkman. In other words, C57BL / 6J mice were anesthetized with 70% N 2 O, 30% O 2 gas and 1.5% isoflurane, and the skin of the forehead was incised. The muscles were opened to expose the trachea. A little incision was made. Using a 1 ml syringe equipped with a rounded 19 gauge needle, 50 μl of the distilled water solution, in which bromomycin was dissolved, was directly injected into the lungs through the incision. Immediately after the injection, the skin of the incisional incision was sutured and awakened from the anesthesia, and then housed in a general breeding cage. The administration of bleomycin was performed using a video instillobot and 40 μg / 50 μl of bleomycin-HCl was administered once to induce pulmonary fibrosis for 12 days.

유파틸린은 DPBS buffer(1% DMSO 함유)에 녹여 사용하였으며, 유파틸린의 투여액량은 1㎖/㎏으로 하였고, 개체별 투여량은 최근 체중 측정을 기준으로 산출하였다. 블레오마이신 투여 12일 후, 마이크로 피펫을 이용하여 1일 1회(주 5회), 1주간 강제 비강투여하였다. 유파틸린 투여 후 2 ~ 3일간의 독성증상 및 사망유무를 관찰하였으나, 블레오마이신과 유파틸린을 투여한 후 별다른 이상증상이 관찰되지 않았다.The tiline was dissolved in DPBS buffer (containing 1% DMSO). The dose of tiline was 1 ml / kg, and individual dose was calculated based on the recent body weight measurement. Twelve days after the administration of bleomycin, the mice were given a forced nasal cavity for one week with a micropipette once a day (five times a week). We observed toxic symptoms and deaths for 2 ~ 3 days after the administration of yupatiline, but no abnormal symptoms were observed after administration of bleomycin and yupatiline.

각 실험군 당 3마리를 선정하여 폐조직을 분리하였다. 분리된 폐조직을 masson's trichrome staining하여 현미경으로 관찰한 결과, 블레오마이신의 처리로 폐섬유화가 유도되었으며, 유파틸린 20㎍ 투여군 및 40㎍ 투여군에서 유파틸린의 투여로 폐섬유화가 저해되는 것을 확인할 수 있었다. 특히 유파틸린 40㎍ 투여군에서 폐섬유화의 보다 효과적인 저해를 확인할 수 있었다(도 6 참조).Three mice were selected per each experimental group and lung tissue was isolated. The results of microscopic examination of the isolated lung tissue with masson's trichrome showed that pulmonary fibrosis was induced by treatment with bleomycin, and that in the 20 ㎍-treated group and 40 유-treated group, . Particularly, in the group administered with 40 쨉 g of tilapine, more effective inhibition of pulmonary fibrosis was confirmed (see Fig. 6).

3-2. 유파틸린을 이용한 ONGHEPA1의 이용 가능성 검증3-2. Validation of availability of ONGHEPA1 using the tiline

상기와 같이 섬유증 치료 효과가 확인된 유파틸린을 이용하여 섬유증 치료제 스크리닝에서 ONGHEPA1의 이용 가능성을 검증하였다. ONGHEPA1의 섬유화가 유도되는 조건에서 유파틸린에 의해 섬유화가 억제된다면 이는 ONGHEPA1을 섬유증 치료제 스크리닝을 위한 세포로 이용할 수 있다는 것을 의미한다.We confirmed the availability of ONGHEPA1 in the screening of fibrosis treatment with the use of empirical tillin, which has been shown to be effective in treating fibrosis as described above. If fibrosis is suppressed by the presence of the gene in the presence of ONGHEPA1 fibrosis, this means that ONGHEPA1 can be used as a cell for the screening of fibrosis therapeutic agents.

이에 ONGHEPA1을 배양하면서 TGF-β(5ng/㎖) 또는 PDGF(5ng/㎖)를 처리하여 섬유화를 유도하되, 배양액 상에 50μM의 농도로 유파틸린을 첨가하였다. 이의 결과 유파틸린을 처리한 실험군은 TGF-β 또는 PDGF를 처리한 섬유화 실험군에서와는 달리 24시간 이후에 HSC가 근섬유아세포(myofibroblast)로 분화되는 현상 및 섬유화 현상이 관찰되지 않았다(도 7 참조).TGF-β (5 ng / ㎖) or PDGF (5 ng / ㎖) was treated with ONGHEPA1 to induce fibrosis, but the concentration of 50 쨉 M was added to the culture solution. As a result, unlike fibrosis treated group treated with TGF-β or PDGF, the experimental group treated with oil tillin did not show the fibrosis phenomenon and the differentiation of HSC into myofibroblast after 24 hours (see FIG. 7).

상기와 같은 실험을 검증하기 위해, ONGHEPA1이 아닌 NHLF(normal human lung fibroblast)을 이용하여 동일한 방법으로 실험하였다. NHLF는 특발성 섬유화의 원인으로 알려져 있다. 이의 결과, 도 8과 같이 NHLF 또한 ONGHEPA1에서와 같은 경향을 나타내었다.In order to verify the above experiment, the same experiment was carried out using NHLF (normal human lung fibroblast) instead of ONGHEPA1. NHLF is known to be the cause of idiopathic fibrosis. As a result, as shown in FIG. 8, NHLF also showed the same tendency as in ONGHEPA1.

실시예 4. ONGHEPA1의 유지 및 관리Example 4. Maintenance and management of ONGHEPA1

4-1. 기자재 및 소모품4-1. Equipment and consumables

4-1-1. 장비4-1-1. equipment

Biosafety cabinet(Ex. BL2, SterilGard Hood, The Baker Company), Pipet-aid(Drummond), 원심분리기(Ependorf), 37℃ 항온수조, CO2 incubator(Thermo), Hemocytometer, Phase-contrast microscopy(Olympus CKX31), Freezing container(cryobox, 예, Nalgene), 초저온 냉동고(Thermo -80℃), Nitrogen tank(예, MVE500)(Biochemistry), Biosafety cabinet (Ex. BL2, SterilGard Hood, The Baker Company), Pipet-aid (Drummond), Centrifuge (Ependorf), 37 ° C water bath, CO 2 incubator Freezing container (cryobox, eg Nalgene), cryogenic freezer (Thermo-80 ℃), nitrogen tank (eg MVE500)

4-1-2. 소모품4-1-2. Expendables

Polypropylene Conical Tube(예, BD FalconTM), 70% Ethanol이 든 스프레이, Serological Pipet, Cryovial(예, Nunc cryo freezing vial cat no. 377267), 라벨 마커 및 라벨, 드라이아이스와 스티로폼 박스Polypropylene Conical Tube (eg BD Falcon ™), spray with 70% Ethanol, Serological Pipet, Cryovial (eg Nunc cryo freezing vial cat no. 377267), label marker and label, dry ice and styrofoam box

4-1-3. 배지 및 일반시약4-1-3. Medium and general reagents

DMEM high glucose(Hyclone, US), Penicillin streptomycin(Hyclone, US), Fetal Bovine Serum(FBS)(Hyclone, US), Dulbesco phosphate Buffered Saline, Trypan blue solution 0.4%(in normal saline)(Gibco, Cat No. 15250-061)(세포수 계수를 위해 사용), DMSO(Sigma-aldrich, Cat no. D2650)(동결보존을 위해 사용), Trypsin/EDTA(Gibco, Cat no. 25300)(세포의 분리를 위해 사용)(Hyclone, US), Penicillin streptomycin (Hyclone, US), Fetal Bovine Serum (FBS) (Hyclone, US), Dulbesco phosphate buffered saline, Trypan blue solution 0.4% (in normal saline) (Gibco Cat. (Used for cell counting), DMSO (Sigma-aldrich, Cat no. D2650) (used for cryopreservation), Trypsin / EDTA (Gibco, Cat No. 25300) )

예) 배양액의 제조(500㎖ 기준) : DMEM high glucose(500㎖) + FBS(50㎖) + 100U/㎖ penicillin/streptocmycin(5.5㎖)Example) Preparation of culture medium (based on 500 ml): DMEM high glucose (500 ml) + FBS (50 ml) + 100 U / ml penicillin / streptocmycin (5.5 ml)

4-2. 동결된 ONGHEPA1의 해동4-2. Thawing of frozen ONGHEPA1

Cryovial 내에 동결된 ONGHEPA1의 해동방법은 다음과 같다.The thawing method of ONGHEPA1 frozen in cryovial is as follows.

사전준비 : 세포희석액을 미리 준비해 두고(예; 50㎖ conical tube에 35㎖ 정도의 cooling된 배양액을 넣어 둠), 항온수조를 미리 예열해 둔다.PREPARATION: Prepare a cell dilution solution (eg, 35 mL of cooled culture medium in a 50 mL conical tube), and preheat the incubation bath beforehand.

(1) 액화질소 탱크에서 드라이아이스가 담긴 스티로폼박스에 세포 동결바이알(cryovial)을 옮겨 담는다.(1) Transfer cell freezing cryovials to a styrofoam box containing dry ice in a liquefied nitrogen tank.

(2) Cryovial의 뚜껑(cap)을 잡고 37℃ 항온수조에 뚜껑 부분이 잠기지 않도록 약 20~25초 이내로 담근다. 다수의 cryovial을 처리할 경우 부유용 랙(floating rack)을 이용해도 되나, 해동하면서 바이얼 내부에 형성되는 iceball이 다 녹기 전에 cryovial을 처리할 수 있는 정도의 숫자로 제한한다. 부분 해동된 vial을 흔들거나 뒤집지 않는다.(2) Hold the cap of the cryovial and immerse it in the constant temperature water bath at 37 ℃ for about 20 ~ 25 seconds so as not to lock the lid part. Floating racks can be used to treat multiple cryovials, but the number of iceballs formed inside the vials is limited to the extent that cryovials can be treated before they melt. Do not shake or flip the partially thawed vial.

(3) 신속하게 부분 해동된 vial의 외부를 70% 에탄올스프레이를 뿌린 후, 깨끗한 종이수건으로 닦는다.(3) Spray 70% ethanol spray on the outside of vial which is partially thawed quickly, and clean with clean paper towel.

(4) 크린벤치 내부에서 cryovial의 뚜껑을 열고 2㎖ selogical pipet이나 1㎖ 블루팁 파이펫으로 0.5~1㎖의 배양액을 추가한 후, 미리 준비해둔 배양액이 담겨있는 50㎖ conical tube에 옮긴다. 여기에 배양액으로 50㎖까지 채운다(약 0.5~1㎖의 배양액을 추가하여 cryovial에 잔류한 세포를 회수할 수도 있다).(4) Open the lid of the cryovial inside the clean bench and add 0.5-1 ml of the culture with a 2 ml selogical pipet or 1 ml blue tip pipet, and transfer it to a 50 ml conical tube containing the prepared culture. Fill up to 50 ml with the culture medium (it is also possible to recover the cells remaining in the cryovial by adding about 0.5 to 1 ml of the culture solution).

(5) 250~300xg(약 1200~1300rpm)의 속도로 상온에서 5분간 원심분리 한다.(5) Centrifuge at room temperature for 5 minutes at a speed of 250 to 300 x g (about 1200 to 1300 rpm).

(6) 상층액을 vacuum suction을 이용하여 제거하거나 또는 직접 제거한다(vacuum suction을 이용하는 경우 세포 pallet이 손실되지 않도록 주의).(6) Remove the supernatant by vacuum suction or remove it directly (be careful not to lose the cell pallet when vacuum suction is used).

(7) 세포 침전에 5~20㎖의 배양액(㎖당 대략 2x106 세포의 농도)을 추가하고 파이펫으로 부드럽게 부유시킨다.(7) Add 5 to 20 ml of the culture solution (concentration of approximately 2x10 6 cells per ml) to cell precipitation and float gently with a pipette.

(8) Hematocytometer를 이용하여 세포수를 측정한다.(8) Count the cells using a hematocytometer.

(9) 따뜻한 배양액이 담겨있는 세포배양용 T-75 flask 또는 dish에 세포를 접종한다.(9) Inoculate cells into T-75 flask or dish for cell culture containing warm culture medium.

(10) 37℃에서 5% CO2 인큐베이터에서 24시간 배양 한 다음, 배양액을 교체하여 비부착 세포들 및 세포 debris들을 제거하였다.(10) After incubation at 37 ° C in a 5% CO 2 incubator for 24 hours, the non-adherent cells and cell debris were removed by replacing the culture medium.

(11) 3~4일 간격으로 배양액을 교체해준다.(11) Replace the culture medium every 3 to 4 days.

4-3. ONGHEPA1의 계대배양4-3. Subculture of ONGHEPA1

(1) 배양 7일째, 현미경(phase microscope)으로 세포의 밀도가 배양판에 80% 이상 증식한 것이 확인되면 배양액을 버리고 DPBS(5㎖)를 이용하여 2회 세척 후 vacuum suction을 이용하여 제거한다.(1) On the 7th day after culturing, if it is confirmed by the microscope that the cell density is increased to 80% or more on the culture plate, the culture medium is discarded, washed twice with DPBS (5 ml), and then removed by vacuum suction .

(2) 0.05% trypsin/EDTA(Gibco) 2㎖를 첨가하여 37℃에서 3~5분간 incubation 시킨다.(2) Add 2 ml of 0.05% trypsin / EDTA (Gibco) and incubate at 37 ° C for 3-5 minutes.

(3) 세포의 분리상태를 확인하여 trypsin/EDTA와 동량의 배양액을 넣고 파이펫을 이용하여 세포를 회수한 후 conical tube에 담고 250~300xg(약 1200~1300rpm)의 속도로 3분간 원심분리한다.(3) Confirm separation of cells and incubate the cells in the same volume as trypsin / EDTA. Collect cells in a conical tube using a pipette and centrifuge at a speed of 250 ~ 300xg (about 1200 ~ 1300rpm) for 3 minutes .

(4) Vacuum suction을 이용하여 상층액을 조심스럽게 제거하고, 따뜻한 배양액 8㎖를 conical tube에 넣어 파이펫을 이용해 세포 pallet을 현탁시킨다.(4) Carefully remove the supernatant using Vacuum suction, add 8 ml of warm culture medium to the conical tube, and suspend the cell pallet using a pipette.

(5) 4개의 75T-flask에 따듯한 배양액(18㎖)을 넣고 (4)에서 만들어진 세포현탁액을 2㎖씩 flask에 넣어준다(세포계대 비율은 1:4~5정도로 함).(5) Add 4 ml of warm culture medium (18 ml) to each 75T-flask, and add 2 ml of the cell suspension prepared in (4) to the flask (cell ratio is 1: 4 ~ 5).

(6) 37℃에서 5% CO2 인큐베이터에서 배양한다.(6) Incubate in a 5% CO 2 incubator at 37 ° C.

4-4. ONGHEPA1의 동결보존4-4. Cryopreservation of ONGHEPA1

사전준비 : Cryovial(Nunc cryo freezing vial cat no. 377267), 라벨 마커 및 라벨, Freezing container(cryobox, 예, Nalgene) 및 동결보존액(보체-비활성화 처리한 FBS:DMSO=9:1)을 준비한다.Prepare: Prepare cryovial (Nunc cryo freezing vial cat no. 377267), label marker and label, freezing container (cryobox, eg Nalgene) and cryoprotectant (complement-deactivated FBS: DMSO = 9: 1).

(1) 배양액을 버리고 DPBS(5㎖)를 이용하여 2회 세척 후 vacuum suction을 이용하여 제거한다.(1) Discard the culture medium, wash it twice with DPBS (5 ml), and remove it by vacuum suction.

(2) 0.05% trypsin/EDTA(Gibco) 2㎖를 첨가하여 37℃에서 3~5분간 incubation 시킨다.(2) Add 2 ml of 0.05% trypsin / EDTA (Gibco) and incubate at 37 ° C for 3-5 minutes.

(3) 세포의 분리상태를 확인하여 trypsin/EDTA와 동량의 배양액을 넣고 파이펫을 이용하여 세포를 수집 후 conical tube에 담고 250~300xg(약 1200~1300rpm)의 속도로 3분간 원심분리한다.(3) Confirm cell separation and incubate with trypsin / EDTA in the same volume. Pipette the cells and collect them in a conical tube. Centrifuge at 250 ~ 300xg (about 1200 ~ 1300rpm) for 3 minutes.

(4) Vacuum suction을 이용하여 상층액을 조심스럽게 제거하고, 37℃에서 데워진 냉동보존액 1㎖(세포수에 따라 보존액은 증량할 수 있음)를 conical tube에 넣고 파이펫을 이용해 세포를 재부유시킨다(일반적으로 배지 1㎖ 당 5~10x106개의 세포를 부유).(4) Carefully remove the supernatant using vacuum suction, add 1 ml of frozen stock (warmed at 37 ° C) to the conical tube, and re-suspend the cells using a pipette (Typically 5-10 x 10 < 6 > cells suspended per 1 ml of medium).

(5) 부유 즉시 pipet으로 부유된 세포를 사전 라벨된 cryovial에 분주한 다음, isopropanol이 충진되어 있는 cyrobox에 넣어 -80℃ 냉동고에 옮겨 보관한다.(5) Float Immediately pipet the cells suspended in the pipet into pre-labeled cryovials, place in a cyrobox filled with isopropanol and store in a -80 ° C freezer.

(6) -80℃ 냉동고에서 최소 3시간 또는 최대 1주일 이내 보관된 세포들은 장기보존을 위해 액화질소저장기의 증기상(vapor phase)으로 이송한다.(6) Cells stored in a -80 ° C freezer for at least 3 hours or up to 1 week are transferred to the vapor phase of the liquefied nitrogen reservoir for long-term storage.

실시예 5. ONGHEPA1를 이용한 스크리닝Example 5. Screening with ONGHEPA1

5-1. 방법5-1. Way

사전준비 : Serum이 포함된 DMEM high glucose, Serum이 포함되지 않은 DMEM high glucose, Recombinant human TGFb-1(10㎍, Peprotech), 유파틸린(5㎎, Adipogen), DPBS, 24 well plate 또는 6 well plate, DMSOPreparation: Serum-containing DMEM high glucose, DMEM high glucose without serum, recombinant human TGFb-1 (10 μg, Peprotech), milk tilline (5 mg, Adipogen), DPBS, 24 well plate or 6 well plate , DMSO

본 실시예에서 사용되는 약물의 최종 농도The final concentration of the drug used in this example

섬유화 유도를 위한 hTGFβ-1의 농도: 5ng/㎖Concentration of hTGF beta-1 for induction of fibrosis: 5 ng / ml

섬유화 억제를 위한 유파틸린 및 스크리닝 화합물의 농도: 50μM/㎖Concentration of flow tiller and screening compound for inhibition of fibrosis: 50 [mu] M / ml

hTGFβ-1의 용해는 serum이 첨가되지 않은 배양액을 사용Dissolution of hTGFβ-1 was performed using culture medium without serum

유파틸린 및 스크리닝 화합물의 용해는 DMSO를 사용Dissolution of the flow tiller and screening compound using DMSO

5-1-1. Serum condition5-1-1. Serum condition

(1) 24 well plate를 이용할 경우 10% FBS, 1% penicillin streptomycin이 포함된 DMEM 배지에 약 4x104세포를 seeding한 후, 37℃, 5% CO2 incubator에서 배양(1) When using a 24-well plate, approximately 4 × 10 4 cells were seeded in a DMEM medium containing 10% FBS and 1% penicillin streptomycin, and cultured at 37 ° C. in a 5% CO 2 incubator

(2) 배양판에 약 50~60%의 세포가 차게 되면(초기 seeding 후 2일 정도 경과) 실험을 시작(serum이 있는 경우에는 시간이 지나면서 세포가 꾸준히 증식하기 때문에 과잉증식으로 인해 좋은 결과를 얻을 수 없음).(2) When about 50-60% of the cells are incubated in the culture plate (about 2 days after the initial seeding), the experiment is started (when the serum is present, the cells proliferate steadily over time, Can not be obtained).

(3) 배양액을 vacuum suction을 이용해 제거하고 DPBS를 사용하여 2~3회 세척(3) The culture solution was removed by vacuum suction and washed 2 ~ 3 times with DPBS

(4) 유파틸린 또는 스크리닝 화합물이 용해된 배양액(serum 포함) 1㎖를 각 well에 넣어줌(4) Add 1 ml of the culture solution (including serum) in which the tiline or screening compound is dissolved in each well

(5) 유파틸린 또는 스크리닝 화합물 처리 12 시간, 24 시간 후 세포형태 확인을 위한 사진촬영 및 RNA 수집(serum 조건에서는 섬유화 속도가 serum free에서 보다 빠르게 일어나기 때문에 12시간을 확인해 볼 필요가 있음)(5) Photographing and RNA collection for confirmation of cell morphology after 12 hours and 24 hours of treatment with either tiline or screening compound (12 hours should be checked because the rate of fibrosis occurs more rapidly in serum free conditions)

5-1-2. Serum free condition5-1-2. Serum free condition

(1) 24 well plate를 이용할 경우 10% FBS, 1% penicillin streptomycin이 포함된 DMEM 배지에 약 1x105세포를 seeding한 후, 37℃, 5% CO2 incubator에서 배양(1) When using a 24-well plate, approximately 1 × 10 5 cells were seeded in DMEM medium containing 10% FBS and 1% penicillin streptomycin, and cultured at 37 ° C. in a 5% CO 2 incubator

(2) 2일 정도 지나면 배양판에 약 80%의 세포가 차게 되며, 이 시점에서 테스트 실시(만일 4x104의 세포를 seeding 했다면 3~4일 후에 80% 정도 배양판에 차게 됨, serum이 없는 실험의 경우 초기에 seeding 하는 세포를 증가시키는 것이 좋음)(2) After about 2 days, about 80% of the cells will become cold on the culture plate. At this point, a test is carried out (if seeding 4x10 4 cells, In the case of experiments, it is better to increase the number of seeding cells initially)

(3) 배지를 vacuum suction을 이용하여 제거하고 DPBS를 이용하여 3회 정도 세척하여 잔류 FBS가 남지 않도록 함(FBS가 소량이라도 남아있는 경우 약제의 효과에 영향을 줄 수 있음)(3) Remove the medium by vacuum suction and wash it three times with DPBS so that no residual FBS is left (it may affect the effect of the drug if a small amount of FBS remains)

(4) 유파틸린 또는 스크리닝 화합물이 용해된 serum free 배지 1㎖을 각 well에 주입(4) Infusion of 1 ml of serum-free medium in which oil tiline or screening compound is dissolved is injected into each well

(5) 유파틸린 또는 스크리닝 화합물 처리 6, 12, 24, 48 시간 후 세포 형태 확인을 위해 사진촬영 및 RNA 수집(5) After 6, 12, 24, and 48 hours of treatment with either tiline or screening compound, photographs and RNA collection

5-2. 결과5-2. result

상기 실시예 5-1의 방법을 이용하여 항섬유화 효과가 있는 화합물을 스크리닝하였다. 이의 결과, 여러 화합물들 중 유파틸린을 포함하여 ONGE200[5,7-다이하이드록시-2-(4-하이드록시페닐)-6-메톡시-크로몬]이 항섬유화 효과가 우수한 것으로 나타났다(도 9 및 10 참조). ONGE200의 화학구조는 다음 화학식 2와 같다.A compound having an anti-fibrotic effect was screened using the method of Example 5-1. As a result, ONGE200 [5,7-dihydroxy-2- (4-hydroxyphenyl) -6-methoxy-chromone] among various compounds was found to be excellent in antifibrotic effect including oil tillin 9 and 10). The chemical structure of ONGE200 is shown in the following chemical formula 2.

[화학식 2](2)

Figure pat00002
Figure pat00002

실시예 6. EMT 관련 유전자의 발현 양상 분석Example 6. Analysis of Expression Pattern of EMT-Related Gene

ONGHEPA1에서 섬유화가 유도되는 조건 및 유파틸린 또는 ONGE200에 의해 섬유화가 억제되는 조건에서 EMT 관련 유전자의 발현 양상을 조사하기 위하여, TGF-β 또는 PDGF를 처리한 ONGHEPA1(대조군)(섬유화 유도) 및 TGF-β 또는 PDGF와 유파틸린 또는 ONGE200을 함께 처리한 ONGHEPA1(실험군)(섬유화 억제)으로부터 total RNA를 정제하여 real-time PCR을 통해 EMT 관련 유전자 발현 양상을 분석하였다. 이의 결과, 도 11에서와 같이 유파틸린 또는 ONGE200에 의해 EMT 관련 유전자들의 발현이 저해되는 것으로 나타났다.In order to investigate the expression pattern of EMT-related genes in ONGHEPA1 induced fibrosis and fibrosis inhibited by ONG200, ONGHEPA1 (control) (induced fibrosis) and TGF- Total RNA was purified from ONGHEPA1 (experimental group) (inhibition of fibrosis), which was treated with β or PDGF and either pravastatin or ONGE200, and the expression pattern of EMT-related gene was analyzed by real-time PCR. As a result, as shown in Fig. 11, the expression of EMT-related genes was inhibited by either tiline or ONGE200.

상기와 같은 실험을 검증하기 위해, ONGHEPA1이 아닌 NHLF을 이용하여 동일한 방법으로 실험하였다. 이의 결과, 도 12에서와 같이 NHLF 또한 ONGHEPA1에서와 같은 경향을 나타내었다.In order to verify the above experiment, the same experiment was conducted using NHLF instead of ONGHEPA1. As a result, as shown in FIG. 12, NHLF also showed the same tendency as in ONGHEPA1.

상기와 같은 실험결과는 EMT 관련 유전자의 조절을 통해 항섬유화 효과를 나타내는 화합물의 스크리닝에 ONGHEPA1이 매우 효과적이라는 것을 나타낸다.These results indicate that ONGHEPA1 is highly effective in screening compounds that exhibit anti-fibrosis effects through regulation of EMT-related genes.

실시예 7. 글로벌 유전자 발현 양상 분석Example 7. Global gene expression pattern analysis

ONGHEPA1의 섬유화 유도 조건 및 섬유화 억제 조건에서 보다 다양한 유전자의 발현 양상을 살펴보기 위하여, DMSO를 처리한 ONGHEPA1(대조군), TGF-β의 처리로 인해 섬유화가 유도된 ONGHEPA1 및 TGF-β와 유파틸린을 함께 처리한 ONGHEPA1의 글로벌 유전자 발현 양상을 조사하였다.To examine the expression patterns of various genes in ONGHEPA1 fibrosis inducing and fibrosis inhibiting conditions, ONGHEPA1 (control group) treated with DMSO, ONGHEPA1 and TGF-β induced by fibrosis induced by treatment of TGF-β, The global gene expression pattern of ONGHEPA1 treated together was investigated.

DMSO, TGF-β 또는 유파틸린을 각각 처리하고 24시간 후에 total RNA를 분리하여 라이브러리를 제작한 다음 Illumina High-seq 시퀀서로 30 Giga의 전체 transcriptome 약 10,000여 개의 발현된 mRNA를 분석하였다.DMSO, TGF-? Or? -Fatilin, respectively. After 24 hours, the total RNA was isolated and the library was prepared. Then, approximately 10,000 transcriptomic mRNAs of 30 Giga total transcriptome were analyzed by Illumina High-seq sequencer.

이의 결과, 다음과 같은 결과가 나타났다(도 13 참조).As a result, the following results were obtained (see FIG. 13).

1) 3가지 실험군에 공통으로 발현되는 유전자 수 : 9,033개1) Number of genes commonly expressed in three experimental groups: 9,033

2) 대조군 ONGHEPA1 세포주에 특이적으로 발현하는 유전자 : 628개2) genes specifically expressed in the control ONGHEPA1 cell line: 628

3) TGF-β 처리 시 특이적으로 발현하는 유전자 : 169개3) Genes specifically expressed in TGF-β treatment: 169

4) TGF-β와 유파틸린 처리 시 특이적으로 발현되는 유전자 : 150개4) TGF-β and genes specifically expressed in the treatment with milk tilline: 150

5) TGF-β 처리군 및 TGF-β+유파틸린 처리군에서 공통으로 발현되는 유전자 : 657개5) Genes commonly expressed in TGF-beta and TGF-beta +

6) 대조군 및 TGF-β 처리군에서 공통으로 발현되는 유전자 : 171개6) Genes Commonly Expressed in Control and TGF-β Treated Group: 171

7) 대조군 및 TGF-β+유파틸린 처리군에서 공통으로 발현되는 유전자 : 188개7) Genes Commonly Expressed in Control and TGF-β +

이에 따라, ONGHEPA1 세포주에 TGF-β를 처리하면 826(=169 + 657)개 유전자의 발현이 유도되어 EMT program이 활성화되고 여기에 유파틸린을 처리하면 328(=150+188)개 유전자의 발현이 다시 유도되어 EMT program을 본래의 상태로 역전시키는 trans-differentiation이 발생한다는 것을 알 수 있다.Thus, treatment of ONGHEPA1 cell line with TGF-β induces the expression of 826 (= 169 + 657) genes and activates the EMT program. When treated with taphyline, the expression of 328 (= 150 + 188) And the trans-differentiation in which the EMT program is reversed to the original state occurs again.

TGF-β 처리군 및 TGF-β+유파틸린 처리군에서 발현이 변화되는 유전자의 유전자 induction fold와 p-value를 비교하여 도 14와 같은 volcano plot으로 나타내었다.The gene induction folds and p-values of the genes whose expression was changed in the TGF-beta treated group and the TGF-beta + yaffa tilin treated group were compared with each other by the volcano plot shown in Fig.

EMT는 종양 발생, 줄기세포 분화 및 섬유화에 관여되는 cellular program으로 현재까지 수백여 개의 유전자가 관여한다는 것이 알려져 있다. Col11a1(Collagen type XI alpha 1), Postn(Periostin), Slit3(Slit homolog 3), Fn1(Fibronectin 1), Axl(AXL receptor tyrosine kinase), Aurka(Aurora kinase A) 등이 EMT marker들이다. 상기 실시예 6에서 유파틸린이 TGF-β에 의한 Col11a1, Postn, Slit3, Fn1, Aurka 유전자의 발현을 억제하는 것으로 나타났고, RNA-Seq 결과를 보면 976개의 유전자가 TGF-β 또는 유파틸린 처리에 의하여 새롭게 발현되는 것으로 나타났으며, 모든 처리에서 공통으로 발견되는 9,033개의 유전자 발현도 미시적으로 발현 증감이 있어서 이들 모두 직, 간접적으로 EMT에 영향을 미칠 것이라 판단된다. 따라서 TGF-β+유파틸린에 처리에 의하여 통계적으로 p<0.05 이하로 발현이 조절되는 이들 10,000여 개의 유전자들을 모두 선택하고 선택된 유전자 각각에 대해 유전자가 코딩하는 단백질의 모든 기능을 통합한 big data 기반의 유전자 interactome을 unbiased manner로 분석하여 이들 간의 네트워크를 조사해 보았다(도 15 참조).EMT is a cellular program involved in tumorigenesis, stem cell differentiation, and fibrosis, and it is known that several hundred genes are involved up to now. These are EMT markers such as Col11a1 (Collagen type XI alpha 1), Postn (Periostin), Slit3 (Slit homolog 3), Fn1 (Fibronectin 1), Axl (AXL receptor tyrosine kinase) and Aurka (Aurora kinase A). In Example 6, it was shown that the expression of Col11a1, Postn, Slit3, Fn1 and Aurka genes was suppressed by TGF-β and that the RNA-Seq results showed that 976 genes were inhibited by TGF- , And 9,033 gene expressions commonly found in all treatments are also microscopically expressed and decreased, and these factors directly or indirectly affect EMT. Thus, TGF-β + and / or taphylline are statistically regulated below p <0.05 by selecting all of these 10,000 genes. (See Figure 15).

놀랍게도 이들 유전자 네트워크의 허브(hub)는 8개로 여러 노드(nod)를 매개하여 하나의 네트워크 프레임워크를 구성하며, 8개의 허브가 대부분 EMT에 관련된 유전자로 구성 되어 있는 것으로 나타났다. 세포골격 단백체 허브(I) 및 콜라젠 단백체 허브(II)는 integrin α1(Itga1) 노드에 의하여 네트워크가 형성되며, EMT의 중요인자로 알려진 Cyclin B1을 포함한 cell cycle 단백체 허브-1(III)은 protein kinase C alpha(PKCA) 노드에 연결되어 위 3개 단백체 허브와 함께 integrin beta 3(Itbg3) 노드에 의하여 견고한 네트워크를 구축하고 있는 것으로 나타났다. 다른 두 번째 cell cycle 허브-2(IV)는 전사인자 Lymphoid Enhancer Binding Factor 1(Lef1) 노드가 Cyclin D1(Ccdn1)에 의해 연결되고 Adenylate Cyclase 9(Adcy9) 신호전달 허브가 EMT에 중요한 키모카인 CXCL16을 매개로 EMT 인자들 C-Fos, CCL2, Junb와 네트워크로 연결 되어 있는 것으로 나타났다. 여기서 Adcy9 신호전달 허브는 유파틸린에 의하여 영향을 받는 새로운 EMT regulator라고 사료된다. 암세포의 EMT에 지대한 영향을 주는 것으로 알려진 CD44 허브(V)는 cell migration 및 invasion에 관여하는 secreted phosphoprotein 1(Spp1/Osteopontin) 노드와 연결되어 있는 것으로 나타났다. Spp1(osteopontin) 역시 중요한 EMT 인자로 알려져 있다. Spp1 노드는 ECM protease인 Mmp3와 네트워크로 연결되어 있었다. ECM matrix의 중요 단백질이고 EMT 주요 인자인 Fibrillin 및 Elastin을 포함한 허브(VI)는 central 노드인 integrin b3(Itgb3)에 연결되어 있는 것으로 나타났다. EMT 원인 사이토카인 transforming growth factor b2 단백체 허브(VII)는 serpine1 및 Figf(=VegfD)를 포함하고 kinase insert domain receptor(Kdr) 노드와 네트워크를 구축하고 있는 것으로 나타났다. EMT의 중요 수용체로 알려진 semaphorin과 콜레스테롤 수용체(Vldr) 허브(VIII)에는 plexin D, semaphoring 3E, neurophilin 및 NGF를 포함하고 있고 semaphorin 수용체 허브는 Kdr 노드와 네트워크를 형성하고 있는 것으로 나타났다. Vldr 수용체 허브는 nerve growth factor(Ngf)를 통해 insulin receptor substrate 2I(rs2)와 연결되어 신호전달 축이 형성되어 있는 것으로 나타났다. EMT의 주요 전사인자인 Snail2(=Slug)-Ecadherin(=Cadh1) 노드는 cell cycle 단백체 허브(II)와 네트워크를 구축하고 Ecadherin은 중요한 EMT 인자들 Mmp3, caviolin(Cav), Tenascin C(Tnc) 및 PKCa와 연결되어 있는 것으로 나타났다. PKCa는 integrin b4에 의하여 세포골격 및 콜라젠 단백체 허브와 네트워크를 구축하고 있었다. Tenascin C는 integrin b3와 직접 연결 되어 있었다.Surprisingly, the hubs of these gene networks consisted of 8 genes that constitute a network framework by mediating multiple nodes (nods), and 8 hubs were composed mostly of EMT related genes. Cell cycle proteolytic herb-1 (III), including Cyclin B1, which is known to be an important factor of EMT, forms a network by the integrin α1 (Itga1) node in the cytoskeletal protease hub (I) C alpha (PKCA) node, which together with the above three proteomic hubs constitute a robust network by the integrin beta 3 (Itbg3) node. The second cell cycle herb-2 (IV) is a mutation of the Cyclin D1 (Ccdn1) node of the Lymphoid Enhancer Binding Factor 1 (Lef1) node and the Adenocyte Cyclase 9 (Adcy9) And the EMT factors C-Fos, CCL2, and Junb as a mediator. Here, the Adcy9 signaling hub is considered to be a new EMT regulator influenced by the ephedrine. The CD44 herb (V), which is known to exert a great influence on the EMT of cancer cells, is associated with a secreted phosphoprotein 1 (Spp1 / Osteopontin) node involved in cell migration and invasion. Spp1 (osteopontin) is also known to be an important EMT factor. The Spp1 node was networked with Mmp3, an ECM protease. The major protein of the ECM matrix and the main factor of EMT, Fibrillin and Herb (VI) including Elastin, were found to be linked to the central node integrin b3 (Itgb3). The transforming growth factor b2 proteomic hub (VII), which is an EMT-mediated cytokine, contains serpine1 and Figf (= VegfD) and is found to be networked with a kinase insert domain receptor (Kdr) node. Semaphorin and cholesterol receptors (Vldr), known as EMT receptors (VIII), contain plexin D, semaphorin 3E, neurophilin and NGF, and semaphorin receptor hubs are networked with Kdr nodes. The Vldr receptor hub was linked to the insulin receptor substrate 2I (rs2) via nerve growth factor (Ngf) and signaling axis was formed. The major transcription factor of EMT, Snail2 (= Slug) -Ecadherin (= Cadh1) node, establishes a network with the cell cycle proteomic hub (II), Ecadherin is an important EMT factor, Mmp3, caviolin (Cav), Tenascin C PKCa. PKCa was building networks with cytoskeleton and collagen proteome hubs by integrin b4. Tenascin C was directly linked to integrin b3.

< 허브 I. 세포골격 단백체 허브 ><Herb I. Cytoskeletal Protein Hub>

Troponin I1 & Troponin I2, Tropomyosin 2, Transgelin, α2 smooth muscle actin, Myosin heavy chain 9 & 11, Leiomodin 1, γ2 smooth muscle acitin, Laminin subunit α4Troponin I1 and Troponin I2, Tropomyosin 2, Transgelin, α2 smooth muscle actin, Myosin heavy chain 9 & 11, Leiomodin 1, γ2 smooth muscle acitin, Laminin subunit α4

<허브 II. 콜라겐 단백체 허브><Hub II. Collagen Protein Hub>

Collagen 4 α5 & α6, Collagen 5 α1 & α3, Collagen 6 α3, Collagen 8 α1 & α5, Collagen 11 α1, Collagen 12 α1, Collagen 15 α1Collagen 4 α5 & α6, Collagen 5 α1 & α3, Collagen 6 α3, Collagen 8 α1 & α5, Collagen 11 α1, Collagen 12 α1, Collagen 15 α1

<허브 III. Cell cycle 단백체 허브-1><Herb III. Cell cycle Protein Hub-1>

Cyclin B1, Gadd45a, Cyclin F, ASPM, NIMA-related kinase (Nek2), OptineurinCyclin B1, Gadd45a, Cyclin F, ASPM, NIMA-related kinase (Nek2), Optineurin

<허브 IV. Cell cycle 단백체 허브-2><Herb IV. Cell cycle Protein herb-2>

Cyclin D1, Cdk14, C-Fos, Junb, CCL2, CCL7Cyclin D1, Cdk14, C-Fos, Junb, CCL2, CCL7

<허브 V. CD44 연관 단백체 허브>&Lt; Hub V. CD44-associated proteolytic herb &

Cd44, Hypoxia Up-Regulated 1 (Hyou1), Ncam, Calreticulin, Immunity-Related GTPase M (Irgm1), Parp4, Parp9, Pdia4 & Pdia6Cd44, Hypoxia Up-Regulated 1 (Hyou1), Ncam, Calreticulin, Immunity-Related GTPase M (Irgm1), Parp4, Parp9, Pdia4 &

<허브 VI. Fibrillin 단백체 허브><Herbs VI. Fibrillin Protein Hub>

Efemp2 (EGF Containing Fibulin-Like Extracellular Matrix Protein 2), Fibrillin 5 (Fbn5), Fibrillin 2 (Fbn2), Elastin (Eln), Fibrillin 1 (Fbn1)Fibrillin 5 (Fbn5), Fibrillin 2 (Fbn2), Elastin (Eln), Fibrillin 1 (Fbn1), Efemp2 (EGF Containing Fibulin-Like Extracellular Matrix Protein 2)

<허브 VII. Transforming Growth Factor Beta 2 단백체 허브><Herb VII. Transforming Growth Factor Beta 2 Protein Herb>

RAR Related Orphan Receptor A (Rora), Neuronal PAS Domain Protein 2 (Npas2), Serpine1, Transforming Growth Factor Beta 2 (Tgfb2), Vascular Endothelial Growth Factor D (Figf)(Ras), Neuronal PAS Domain Protein 2 (Npas2), Serpine1, Transforming Growth Factor Beta2 (Tgfb2), Vascular Endothelial Growth Factor D (Fig.

<허브 VIII. Semaphorin & Vldr 수용체 단백체 허브><Herb VIII. Semaphorin & Vldr Receptor Protein Hub>

Plexin D1, Semaphorin 3E, Semaphorin 3A, Neuropilin 1, Very Low Density Lipoprotein Receptor, Nerve Growth Factor (Ngf)Plexin D1, Semaphorin 3E, Semaphorin 3A, Neuropilin 1, Very Low Density Lipoprotein Receptor, Nerve Growth Factor (Ngf)

<TGF-β-Eupatilin Interactome의 주요 네트워크 노드><Major network node of TGF-β-Eupatilin interactome>

Integrin α1, Integrin β3, Integrin β4, Protein kinase Cα, Lef1, Slug, Cadherin1 (=E-Cadherin), Adenylate cyclase 9, Spp1 (=Osteopointin), Fibrilin1, Dedicator of cytokinesis 1 (Dock1), Syk2, Notch4 등Integrin α1, Integrin β3, Integrin β4, Protein kinase Cα, Lef1, Slug, Cadherin1 (= E-Cadherin), Adenylate cyclase 9, Spp1 (= Osteopointin), Fibrilin1, Dedicator of cytokinesis 1 (Dock1), Syk2,

상기와 같은 결과는 ONGHEPA1에서 TGF-β 처리로 유도되는 EMT 프로그램이 유파틸린에 의해 역전되며, 이때의 메커니즘은 8개의 EMT 단백체 허브들, 즉 세포골격 단백체 허브(I), 콜라젠 단백체 허브(II), Cell cycle 단백체 허브-1(III), Cell cycle 단백체 허브-2(IV), CD44 연관 단백체 허브(V), Fibrillin 단백체 허브(VI), TGF-β2 단백체 허브(VII), 및 Semaphorin 및 Vldr 수용체 단백체 허브(VIII)의 생성과 소멸로 이루어지며 integrin α1, intergrin β3, protein kinase Cα, Snali2, Kdr, Ecadherin, adenylate cyclase 9이 중요한 노드로 네트워크가 연결된다는 것을 나타낸다.These results indicate that the EMT program induced by TGF-β treatment in ONGHEPA1 is reversed by the ephtaliline, and the mechanism of this is reversed by 8 EMT protein herbs, ie, cytoskeletal protein hub (I), collagen protein hub (II) , Cell cycle protease herb-1 (III), Cell cycle protease herb-2 (IV), CD44-related proteolytic herb (V), Fibrillin protease hub (VI), TGF-β2 protease hub (VII), and Semaphorin and Vldr receptor It is shown that integrin α1, intergrin β3, protein kinase Cα, Snali2, Kdr, Ecadherin and adenylate cyclase 9 are connected to important nodes.

실시예 8. 표적 유전자 분석Example 8. Target gene analysis

상기 실시예 7에서와 같은 글로벌 유전자 발현 양상 조사결과를 바탕으로, ONGHEPA1 세포에서 TGF-β 처리로 발현이 유도되고, 여기에 유파틸린을 처리하였을 경우 발현이 퇴행하는 유전자, 즉 TGF-β 처리군과 유파틸린 처리군 사이에 발현 차이가 나타나는 유전자들 중, TGF-β 처리로 발현이 크게 증가하였다가 유파틸린의 처리로 발현이 거의 이루어지지 않을 정도로 큰 변화를 나타내는 유전자들을 선별하였다.Based on the results of the global gene expression analysis as in Example 7, the ONGHEPA1 cells were induced to express by TGF-β treatment, and when they were treated with the gene, And TGF-.beta., Respectively. The expression levels of TGF-.beta. And TGF-.beta. Were significantly increased.

이의 결과 다음 표 2 및 5와 같은 103개의 유전자가 선별되었다.As a result, 103 genes as shown in the following Tables 2 and 5 were selected.

No.No. Description (Abbreviation)Description (Abbreviation) Log2fcLog2fc p-value p-value 1One Actin, gamma2 (Actg2)Actin, gamma2 (Actg2) -5.83-5.83 0.000050.00005 22 Periostin (Postn)Periostin (Postn) -4.92-4.92 0.000050.00005 33 Collagen, type XI, alpha 1 (Col11a1)Collagen, type XI, alpha 1 (Col11a1) -3.11-3.11 0.000050.00005 44 Fibronectin 1 (Fn1)Fibronectin 1 (Fn1) -infinite-infinite 0.00020.0002 55 Thrombospondin, type I domain containing 7A (Thsd7a)Thrombospondin, type I domain containing 7A (Thsd7a) -5.06-5.06 0.00020.0002 66 TraB domain containing 2B (Trabd2b)The TraB domain containing 2B (Trabd2b) -2.75-2.75 0.0003 0.0003 77 Collagen type XV, alpha 1 (Col5a1)Collagen type XV, alpha 1 (Col5a1) -2.26-2.26 0.000550.00055 88 Slit homolog 3 (Slit3)Slit homolog 3 (Slit3) -3.65-3.65 0.000650.00065 99 Cell migration inducing protein, hyaluronan biding (Cemip)Cell migration inducing protein, hyaluronan biding (Cemip) -2.75-2.75 0.000950.00095 1010 Inhibition beta-A (Inhba)Inhibition beta-A (Inhba) -2.19-2.19 0.00150.0015 1111 Spectrin alpha, erythrocytic 1 (Spta1)Spectrin alpha, erythrocytic 1 (Spta1) -4.01-4.01 0.001650.00165 1212 Exocyst complex component 4 (Exoc4)Exocyst complex component 4 (Exoc4) -2.9-2.9 0.00190.0019 1313 A disintegrin-like and metallopeptidase with thrombospondin type 1 motif 12 (Adamts12)A disintegrin-like and metallopeptidase with thrombospondin type 1 motif 12 (Adamts12) -2.09-2.09 0.00250.0025 1414 Ephrin B2 (Efnb2)Ephrin B2 (Efnb2) -1.92-1.92 0.00380.0038 1515 c-fos induced growth factor (Figf)c-fos induced growth factor (Figf) -2.49-2.49 0.00440.0044 1616 Elastin (Eln)Elastin (Eln) -3.28-3.28 0.005550.00555 1717 Heparan sulfate 6-O-sulfotransferase 2 (Hs6st2)Heparan sulfate 6-O-sulfotransferase 2 (Hs6st2) -3.26-3.26 0.00560.0056 1818 Perlecan (Heparan sulfate proteoglycan2) (Hspg2Perlecan (Heparan sulfate proteoglycan2) (Hspg2 -infinite-infinite 0.00570.0057 1919 Tubulin-specific chaperone d (Tbcd)Tubulin-specific chaperone d (Tbcd) -2.11-2.11 0.005950.00595 2020 Natriuretic peptide receptor 3 (Npr3)Natriuretic peptide receptor 3 (Npr3) -2.82-2.82 0.006750.00675 2121 Serin (or cysteine) peptidase inhibitor, clade F, member 1 (Serpinf1)Serine (or cysteine) peptidase inhibitor, clade F, member 1 (Serpinf1) -1.99-1.99 0.006850.00685 2222 TLC domain-containing protein 2 (Tlcd2)TLC domain-containing protein 2 (Tlcd2) -infinite-infinite 0.00740.0074 2323 Fras 1 related extracellular matrix protein 1 (Frem1)Fras 1 related extracellular matrix protein 1 (Frem1) -2.36-2.36 0.00750.0075 2424 Caldesmon 1 (Cald1)Caldesmon 1 (Cald1) -infinite-infinite 0.00740.0074 2525 Lysyl oxidase-like 2 (Loxl2)Lysyl oxidase-like 2 (Loxl2) -1.93-1.93 0.00780.0078 2626 Tissue inhibitor of metalloproteinase 3 (Timp3)Tissue inhibitor of metalloproteinase 3 (Timp3) -infinite-infinite 0.007850.00785 2727 Collagen, type III, alpha 1 (Col3a1)Collagen, type III, alpha 1 (Col3a1) -infinite-infinite 0.00830.0083 2828 Protein disulfide isomerase associated 6 (Pdia6)Protein disulfide isomerase associated 6 (Pdia6) -1.85-1.85 0.008350.00835 2929 Pleiotrophin (Ptn)Pleiotrophin (Ptn) -2.06-2.06 0.008750.00875 3030 Prostate androgen-regulated mucin-like protein 1 (Parm1)Prostate androgen-regulated mucin-like protein 1 (Parm1) -1.57-1.57 0.012250.01225

No.No. Description (Abbreviation)Description (Abbreviation) Log2fcLog2fc p-value p-value 3131 Dihydropyrimidinase-like 3 (Dpysl3)Dihydropyrimidinase-like 3 (Dpysl3) -infinite-infinite 0.01380.0138 3232 Collagen, type XII, alpha 1 (Col12a1)Collagen, type XII, alpha 1 (Col12a1) -2.02-2.02 0.014350.01435 3333 Crystallin, zeta (quinone reductase)-like 1 (Cryzl1)Crystallin, zeta (quinone reductase) -like 1 (Cryzl1) -infinite-infinite 0.014750.01475 3434 Calumenin (Calu)Calumenin (Calu) -infinite-infinite 0.0150.015 3535 Follistatin-like 1 (Fstl1)Follistatin-like 1 (Fstl1) -infinite-infinite 0.01560.0156 3636 Vinculin (Vcl)Vinculin (Vcl) -infinite-infinite 0.015750.01575 3737 Cyclin D2 (Ccnd2)Cyclin D2 (Ccnd2) -2.29-2.29 0.015850.01585 3838 A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif 2 (Adamts2)A disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif 2 (Adamts2) -2.08-2.08 0.16850.1685 3939 Dysferlin (Dysf)Dysferlin (Dysf) -2.06-2.06 0.017650.01765 4040 Olfactomedin 2 (Olfm2)Olfactomedin 2 (Olfm2) -1.9-1.9 0.018450.01845 4141 Ubiquitin-like modifier activating enzyme 1 (Uba1)Ubiquitin-like modifier activating enzyme 1 (Uba1) -infinite-infinite 0.018550.01855 4242 Leprecan 1 (Lepre1)Leprecan 1 (Lepre1) -1.75-1.75 0.018650.01865 4343 Prosaposin (Psap)Prosaposin (Psap) -infinite-infinite 0.018750.01875 4444 Latent transforming growth factor beta binding protein 1 (Ltbp1)Latent transforming growth factor beta binding protein 1 (Ltbp1) -3.32-3.32 0.019850.01985 4545 Spectrin beta, non-erythrocytic 1 (Sptbn1)Spectrin beta, non-erythrocytic 1 (Sptbn1) -infinite-infinite 0.020.02 4646 Palladin, cytoskeletal associated protein (Palld)Palladine, cytoskeletal associated protein (Palld) -infinite-infinite 0.020050.02005 4747 Protein FAM 53B (Fam53b)Protein FAM 53B (Fam53b) -infinite-infinite 0.020150.02015 4848 Caveolin 1, Caveolae protein (Cav1)Caveolin 1, Caveolae protein (Cav1) -1.76-1.76 0.020250.02025 4949 Nischarin (Nisch)Nischarin (Nisch) -infinite-infinite 0.020750.02075 5050 Fibronectin type III domain containing 1 (Fndc1)Fibronectin type III domain containing 1 (Fndc1) -1.75-1.75 0.021050.02105 5151 Tropomyosin 1, alpha (Tpm1)Tropomyosin 1, alpha (Tpm1) -infinite-infinite 0.021450.02145 5252 Doublecortin-like kinase 1 (Dclk1)Doublecortin-like kinase 1 (Dclk1) -1.54-1.54 0.0230.023 5353 Actin alpha 4 (Actn4)Actin alpha 4 (Actn 4) -infinite-infinite 0.02410.0241 5454 Colony stimulating factor 1 (macrophage) (Csf1)Colony stimulating factor 1 (macrophage) (Csfl) -2.15-2.15 0.025350.02535 5555 Tenascin C (Tnc)Tenascin C (Tnc) -5.1-5.1 0.025750.02575 5656 Intersectin 1 (SH3 domain protein 1A) (Itsn1)Intersectin 1 (SH3 domain protein 1A) (Itsn1) -infinite-infinite 0.02630.0263 5757 Transforming, acidic coiled-coil containing protein 2 (Tacc2)Transforming, acidic coiled-coil containing protein 2 (Tacc2) -infinite-infinite 0.02670.0267 5858 Pleckstrin and sec7 domain containing 3 (Psd3)Pleckstrin and sec7 domain containing 3 (Psd3) -1.43-1.43 0.02750.0275 5959 C-terminal-binding protein 2 (Ctbp2)C-terminal-binding protein 2 (Ctbp2) -infinite-infinite 0.02770.0277 6060 Heat shock protein 90, alpha (cytosolic), class A member 1(Hsp90aa1)Heat shock protein 90, alpha (cytosolic), class A member 1 (Hsp90aa1) -infinite-infinite 0.0290.029

No.No. Description (Abbreviation)Description (Abbreviation) Log2fcLog2fc p-value p-value 6161 Septin2 (Sept2)Septin 2 (Sept2) -infinite-infinite 0.029750.02975 6262 Epidermal growth factor-containing fibulin-like extracellular matrix protein 2 (Efemp2)Epidermal growth factor-containing fibulin-like extracellular matrix protein 2 (Efemp2) -1.66-1.66 0.030050.03005 6363 EH-domain containing 2 (Ehd2)EH-domain containing 2 (Ehd2) -infinite-infinite 0.030250.03025 6464 Coatomer protein complex, subunit gamma 1 (Copg1)Coatomer protein complex, subunit gamma 1 (Copg1) -infinite-infinite 0.030450.03045 6565 v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (Mycn)v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (Mycn) -2.05-2.05 0.0310.031 6666 Lethal giant larvae homolog 1 (Ligi1)Lethal giant larvae homolog 1 (Lig1) -infinite-infinite 0.03310.0331 6767 Interleukin 18 receptor accessory protein (Il18rap)Interleukin 18 receptor accessory protein (Il18rap) -1.69-1.69 0.03320.0332 6868 Willians-Beuren syndrome chromosome reion 17 homolog (Wbscr17)Willians-Beuren syndrome chromosome reion 17 homolog (Wbscr17) -2.83-2.83 0.033250.03325 6969 Collagen type 1 alpha 1 (Col1a1)Collagen type 1 alpha 1 (Col1a1) -1.64-1.64 0.03340.0334 7070 Synaptopodin (Synpo)Synaptopodin (Synpo) -infinite-infinite 0.033750.03375 7171 Integrin beta 5 (Itgb5)Integrin beta 5 (Itgb5) -infinite-infinite 0.03420.0342 7272 Tankyrase, TRF1-interacting ankyrin-related ADP-rebose polymerase 2 (Tnks2)Tankyrase, TRF1-interacting ankyrin-related ADP-rebose polymerase 2 (Tnks2) -infinite-infinite 0.03490.0349 7373 Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (Plod3)Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 3 (Plod3) -2.01-2.01 0.03550.0355 7474 BTAF1 RNA polymerase II, B-TFIID transcription factor-associated (Btaf1)BTAF1 RNA polymerase II, B-TFIID transcription factor-associated (Btaf1) -infinite-infinite 0.03560.0356 7575 Dynein cytoplasmic 1 heavy chain 1 (Dync1h1)Dynein cytoplasmic 1 heavy chain 1 (Dync1h1) -infinite-infinite 0.035650.03565 7676 Aurora kinase A (Aurka)Aurora kinase A (Aurka) -15.3-15.3 0.035950.03595 7777 WNK lysine deficient protein kinase 1 (Wnk1)WNK lysine deficient protein kinase 1 (Wnk1) -infinite-infinite 0.036850.03685 7878 Collagen type VII alpha1 (Col7a1)Collagen type VII alpha1 (Col7a1) -15.2-15.2 0.037150.03715 7979 Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha 1 polypeptide (P4ha1)Procollagen-proline, 2-oxoglutarate 4-dioxygenase (proline 4-hydroxylase), alpha 1 polypeptide (P4 ha1) -1.81-1.81 0.03750.0375 8080 Spectrin repeat containing nuclear envelope 2 (Syne2)Spectrin repeat containing nuclear envelope 2 (Syne2) -infinite-infinite 0.03760.0376 8181 Cell cycle associated protein 1 (Caprin1)Cell cycle associated protein 1 (Caprin1) -infinite-infinite 0.03820.0382 8282 Calreticlin (Calr)Calreticlin (Calr) -1.74-1.74 0.038450.03845 8383 Endoglin (Eng)Endoglin (Eng) -infinite-infinite 0.038950.03895 8484 Microtubule-associated protein 4 (Map4)Microtubule-associated protein 4 (Map4) -infinite-infinite 0.0390.039 8585 rho/rac guanine nucleotide exchange factor (GEF) 2 (Arhgef2)rho / rac guanine nucleotide exchange factor (GEF) 2 (Arhgef2) -infinite-infinite 0.039150.03915 8686 Inositol hexakisphosphate kinase 1 (Ip6k1)Inositol hexakisphosphate kinase 1 (Ip6k1) -infinite-infinite 0.039850.03985 8787 TEA Domain Transcription factor 1 (TEAD1)TEA Domain Transcription factor 1 (TEAD1) -infinite-infinite 0.040050.04005 8888 Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 (Plod2)Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 (Plod2) -1.48-1.48 0.04020.0402 8989 Family with sequence similarity 175, member B (Fam175b)Family with sequence similarity 175, member B (Fam175b) -infinite-infinite 0.04130.0413 9090 ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (Asap1)ArfGAP with SH3 domain, ankyrin repeat and PH domain 1 (Asp1) -infinite-infinite 0.04140.0414

No.No. Description (Abbreviation)Description (Abbreviation) Log2fcLog2fc p-value p-value 9191 Laminin, alpha 4 (Lama4)Laminin, alpha 4 (Lama4) -1.29-1.29 0.041450.04145 9292 Serine (or cysteine) peptidase inhibitor, clade E, member 1 (Serpine 1)Serine (or cysteine) peptidase inhibitor, clade E, member 1 (Serpine 1) -1.78-1.78 0.04190.0419 9393 Importin-4 (Ipo4)Importin-4 (Ipo4) -infinite-infinite 0.042350.04235 9494 Transformation/ transcription domain-associated protein (Trrap)Transformation / transcription domain-associated protein (Trrap) -infinite-infinite 0.0430.043 9595 Surfeit 1 (SURF1)Surfite 1 (SURF1) -infinite-infinite 0.0440.044 9696 Oxysterol binding protein-like 9 (Osbpl9)Oxysterol binding protein-like 9 (Osbpl9) -infinite-infinite 0.04580.0458 9797 Endoplasmic reticulum-golgi intermediate compartment 1 (ERGIC1)Endoplasmic reticulum-golgi intermediate compartment 1 (ERGIC1) -1.26-1.26 0.04650.0465 9898 Ring finger protein 145 (Rnf145)Ring finger protein 145 (Rnf145) -infinite-infinite 0.046650.04665 9999 AXL receptor tyrosine kinase (Axl)AXL receptor tyrosine kinase (Axl) -infinite -infinite 0.0480.048 100100 Latent transforming growth factor beta binding protein 2 (Ltbp2)Latent transforming growth factor beta binding protein 2 (Ltbp2) -infinite-infinite 0.048450.04845 101101 Latent transforming growth factor beta binding protein 4 (Ltbp4)Latent transforming growth factor beta binding protein 4 (Ltbp4) -infinite-infinite 0.04920.0492 102102 Multiple coagulation factor deficiency 2 (Mcfd2)Multiple coagulation factor deficiency 2 (Mcfd2) -1.28-1.28 0.049350.04935 103103 Thymoma viral proto-oncogene 1 (Akt1)Thymoma viral proto-oncogene 1 (Akt1) -infinite-infinite 0.049850.04985

상기 103개 유전자들의 기능 및 관련 연구결과들을 조사한 결과 대부분이 EMT에 관여하는 인자들로 조사되었으며, 특히 Follistatin-like 1(Fstl1) 유전자의 경우, 이를 낙아웃시키면 블레오마이신으로 폐섬유화를 유도하더라도 폐섬유화가 거의 이루어지지 않는다는 연구결과(Dong et al., 2015)가 있었다.Most of the 103 genes were found to be involved in EMT, and in particular, Follistatin-like 1 (Fstl1) gene, if dropped out, would induce pulmonary fibrosis with bleomycin, There was a study (Dong et al., 2015) that almost no fibrosis was observed.

이에 ONGHEPA1을 이용한 섬유증 치료제 스크리닝에 상기와 같은 103개 유전자들의 발현양상 조사를 적용한다면 EMT의 조절을 통해 섬유화를 억제할 수 있는 화합물을 보다 효율적으로 스크리닝할 수 있을 것이다.Therefore, if the expression patterns of 103 genes are applied to the screening of fibrosis therapeutic using ONGHEPA1, compounds capable of inhibiting fibrosis can be more efficiently screened by controlling EMT.

한국생명공학연구원Korea Biotechnology Research Institute KCTC13086BPKCTC13086BP 2016082520160825

Claims (6)

TGF-β(Transforming growth factor beta) 또는 PDGF(Platelet-derived growth factor)의 처리로 섬유화(fibrosis)가 유도되는 것을 특징으로 하는 간성상세포 유래 간엽줄기세포주 ONGHEPA1(KCTC13086BP).The hepatocyte-derived mesenchymal stem cell line ONGHEPA1 (KCTC13086BP) is characterized in that fibrosis is induced by treatment of TGF-beta (Transforming growth factor beta) or PDGF (Platelet-derived growth factor). 제 1항의 ONGHEPA1에 TGF-β 및 PDGF 중에서 선택된 하나의 단백질 및 후보물질을 처리하는 단계; 및
상기 단백질 및 후보물질을 처리한 ONGHEPA1과 후보물질을 처리하지 않은 대조군의 섬유화 정도를 비교하는 단계;를 포함하는 섬유증 치료제의 스크리닝 방법.Treating ONGHEPA1 of claim 1 with a protein selected from TGF-? And PDGF and a candidate substance; And
Comparing the degree of fibrosis of ONGHEPA1 treated with the protein and the candidate substance and the degree of fibrosis of a control group not treated with the candidate substance. 제 2항에 있어서,
상기 스크리닝 방법은 특발성 폐섬유증(idiopathic pulmonary fibrosis), 골수섬유증(myelofibrosis), 간섬유증(liver fibrosis) 및 신장섬유증(kidney fibrosis)으로 이루어진 군 중에서 선택된 섬유증에 대한 치료제를 스크리닝하기 위한 방법인 것을 특징으로 하는 스크리닝 방법.3. The method of claim 2,
The screening method is a method for screening a therapeutic agent for fibrosis selected from the group consisting of idiopathic pulmonary fibrosis, myelofibrosis, liver fibrosis, and kidney fibrosis Lt; / RTI &gt; 제 2항에 있어서,
상기 섬유화 정도는 세포의 형태를 관찰하는 방법을 이용하여 판단하는 것을 특징으로 하는 스크리닝 방법.3. The method of claim 2,
Wherein the degree of fibrosis is determined using a method of observing the morphology of the cells. 제 2항에 있어서,
상기 단백질 및 후보물질을 처리한 ONGHEPA1과 후보물질을 처리하지 않은 대조군의 유전자의 발현양상을 비교하는 단계;를 더 포함하는 것을 특징으로 하는 스크리닝 방법.3. The method of claim 2,
And comparing the expression patterns of genes of ONGHEPA1 treated with the protein and the candidate substance and a control group not treated with the candidate substance. 제 5항에 있어서,
상기 유전자는 Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpinf1, Tlcd2, Frem1, Cald1, Loxl2, Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vcl, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld, Fam53b, Cav1, Nisch, Fndc1, Tpm1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Il18rap, Wbscr17, Col1a1, Synpo, Itgb5, Tnks2, Plod3, Btaf1, Dync1h1, Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Arhgef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, Osbpl9, ERGIC1, Rnf145, Axl, Ltbp2, Ltbp4, Mcfd2 및 Akt1로 이루어진 군 중에서 선택된 하나 또는 둘 이상의 유전자인 것을 특징으로 하는 스크리닝 방법.
6. The method of claim 5,
The gene is selected from the group consisting of Actg2, Postn, Col11a1, Fn1, Thsd7a, Trabd2b, Col5a1, Slit3, Cemip, Inhba, Spta1, Exoc4, Adamts12, Efnb2, Figf, Eln, Hs6st2, Hspg2, Tbcd, Serpinf1, Tlcd2, Frem1, Cald1, , Timp3, Col3a1, Pdia6, Ptn, Parm1, Dpysl3, Col12a1, Cryzl1, Calu, Fstl1, Vc1, Ccnd2, Adamts2, Dysf, Olfm2, Uba1, Lepre1, Psap, Ltbp1, Sptbn1, Palld, Fam53b, Cav1, Nisch, Fndc1 , Tmp1, Dclk1, Actn4, Csf1, Tnc, Itsn1, Tacc2, Psd3, Ctbp2, Hsp90aa1, Sept2, Efemp2, Ehd2, Copg1, Mycn, Ligi1, Il18rap, Wbscr17, Col1a1, Synpo, Itgb5, Tnks2, Plod3, Btaf1, , Aurka, Wnk1, Col7a1, P4ha1, Syne2, Caprin1, Calr, Eng, Map4, Argef2, Ip6k1, TEAD1, Plod2, Fam175b, Asap1, Lama4, Serpine 1, Trrap, SURF1, Osbpl9, ERGIC1, Rnf145, Axl, Ltbp2, Ltbp4, Mcfd2, and Aktl.
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CN110494556B (en) * 2017-04-03 2024-04-30 株式会社钟化 Cell population containing mesenchymal stem cells, method for producing the same, and pharmaceutical composition
KR102219765B1 (en) 2019-11-13 2021-02-24 부산대학교 산학협력단 Composition for inducing reprogramming into stem cell from hepatic stellate cell, and uses thereof
KR20230105143A (en) * 2022-01-03 2023-07-11 서울대학교산학협력단 Information providing method for diagnosing interstitial fibrosis and tubular atrophy(ifta) in kidney allografts

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