KR20160116750A - Method for the production of "Avatarus", an animal model of Sjogren's syndrome through salivary gland tissue transplantation - Google Patents

Abstract

The present invention relates to a method for preparing an animal model of Sjogren's syndrome, and more particularly, to a method for producing Sjogren's syndrome animal model, which comprises transplanting salivary glands isolated from a patient suffering from Sjogren's syndrome and then further injecting peripheral blood mononuclear cells and saliva And a manufacturing method thereof.
An animal model according to the present invention can provide an animal model for personalized treatment and further inject peripheral blood mononuclear cells and saliva cell lines after transplantation of the salivary gland tissue of a patient to produce an animal model of Sjogren syndrome more effectively Therefore, it is expected to contribute greatly to the development of Sjogren's syndrome treatment and treatment methods.

Description

{Producing method of Sjogren's syndrome animal model, " AbartaSS ", via transplanting Salivary Gland}, an animal model of Sjogren's syndrome through salivary gland tissue transplantation,

The present invention relates to a method for preparing an animal model of Sjogren's syndrome, and more particularly, to a method for producing Sjogren's syndrome animal model, which comprises transplanting salivary glands isolated from a patient suffering from Sjogren's syndrome and then further injecting peripheral blood mononuclear cells and saliva And a manufacturing method thereof.

Autoimmune diseases, particularly Sjögren's syndrome, for example, are still clinically significant diseases in humans. Autoimmune disease, as its name implies, destroys itself through its own immune system. Although the pathological mechanisms between the individual types of autoimmune diseases are different, one common mechanism relates to the binding of specific antibodies present (referred to herein as self-reactive antibodies or autoantibodies).

Sjogren's syndrome is a chronic disease in which white blood cells attack the water-producing spleen. Characteristic symptoms are dry eye syndrome and dry eye syndrome caused by lymphatic component infiltration of lacrimal gland and salivary gland. Loss of tears and saliva can result in a characteristic change in the eye's eye (referred to as aqueous tear deficiency or dry corneal conjunctivitis) and a characteristic change in the mouth (which can lead to tooth damage, increased oral infection, difficulty in swallowing, Pain) may be induced. The patient may also have inflammation of the joints (arthritis), muscle inflammation (myositis), nerve inflammation (neuropathy), thyroid inflammation (thyroiditis), kidney inflammation (nephritis), lung inflammation or other parts of the body, Swelling can occur. In addition, patients may experience fatigue and sleep disturbances. Sjogren's syndrome is one of the most common autoimmune diseases that affects about 4 million Americans, mainly in middle-aged women.

Since the treatment of Sjogren's syndrome is a symptomatic therapy, it is necessary to treat it based on the pathological data. The main symptoms of Sjogren's syndrome include dry symptoms (dry mouth and dry eye), fatigue, arthralgia / arthritis, And other systemic symptoms. The most prevalent symptom is dryness, and the association between subjective symptoms and objective tests is known to be weak, and there is no optimal standard for assessing symptoms.

How Sjogren's syndrome causes autoimmune reactions is not yet clear, but some studies report that the virus is involved in the induction of autoimmune responses. For example, Epstein-Barr virus (EBV), which infects infectious mononucleosis, a disease characterized by swollen salivary glands, joint pain and fatigue, specifically triggers an autoimmune response to genetically susceptible individuals Is known to be able to. These viruses impair the salivary glands and attract "immune" lymphocytes to the salivary glands. These lymphocytes contain specific autoantibodies such as rheumatoid factor (RF), antinuclear antibodies, and sgrene-related antigens A and B (or SS RTI ID = 0.0 > SS-B). ≪ / RTI > Autoantibodies to Ro / SS-A and La / SS-B antigens are present in the tears of some Sjogren's syndrome patients and their presence in serum or tears is known to be associated with severe dry eye keratoconjunctivitis, Antibodies against centromere protein B (CENP B) and center-line protein C (CENP C) are also autoantibodies that occur in Sjogren's syndrome. In some studies, 15% of Sjören's syndrome patients have CENP C , And patients with Sjogren's syndrome were reported to have the autoantibody ICA69.

These antibodies can be introduced into the bloodstream and can be measured in blood tests obtained to confirm the diagnosis of Sjogren ' s syndrome. When excessive T cells enter the gland, damage occurs. Under normal circumstances, a cell class called "inhibitory factor cells" can stop the inflammatory process, but when the glands are continuously destroyed, the excessive action of T- There is an abnormal balance between the deficient actions of T-suppressor cells. Rather than the destruction of these cells, functional impairment is regarded as a major mechanism of secretory failure in Sjogren's syndrome.

On the other hand, tailored medicine is also referred to as custom-made medicine or personalized medicine, and the individual's constitution or environment is individually examined to determine suitable treatment methods It means how to treat. In order to implement customized medicine based on genetic information, a biomarker for dielectrics diagnosis, a variety of genome inspection methods, a medical information system which can be obtained by using the biomarker or analyzed / integrated with the genome information obtained by the above- Analysis method, targeting treatment technique to apply the result analyzed by the above analysis method, and the like should be complementarily developed. That is, it is not only necessary to develop a technique for selecting a suitable treatment method for a patient, but also a technique for verifying the selected treatment method must be developed.

In order to realize customized medicine, it is necessary to develop a method of screening drugs suitable for a patient or selecting a therapeutic method. Especially, in immunological diseases such as Sjogren's syndrome as described above, , It is necessary to develop an animal model for preclinical studies that can create an in vivo environment that reproduces biological characteristics in an individual.

Accordingly, the present inventors have conducted research on an animal model for screening a therapeutic agent for Sjogren's syndrome or developing a therapeutic method with excellent therapeutic effect according to individual biomolecule characteristics and transplanting the salivary gland tissue of the patient, The present invention has been accomplished to produce an effective Sjögren syndrome animal model by additionally injecting peripheral blood mononuclear cells (PBMC) and saliva cell lines.

Korean Patent Publication No. 10-2009-0043910

It is an object of the present invention to provide a novel animal model and a manufacturing method thereof for developing new therapeutic agents and therapeutic methods effective for Sjogren ' s syndrome.

In order to achieve the above object, the present invention provides a method for preparing an animal model of Sjogren's syndrome comprising implanting salivary gland tissue isolated from a patient suffering from Sjogren syndrome into a mouse.

In an embodiment of the present invention, the salivary gland tissue may have a volume of 0.1 cm 3 to 2 cm 3.

In one embodiment of the present invention, the salivary gland tissue may be coated with matrigel and implanted.

In one embodiment of the present invention, the salivary gland tissue may be transplanted into the subcutaneous space secured at an upper portion of 2 to 4 cm from the open point, by 0.5 to 2 cm of the upper 2 to 4 cm of the hindpaw .

In one embodiment of the present invention, the method may further comprise implanting salivary gland tissue isolated from a patient with Sjogren's Syndrome, followed by further infusion of peripheral blood mononuclear cells and saliva cell lines isolated from the patient with Sjogren's syndrome.

In one embodiment of the present invention, the peripheral blood mononuclear cells may be injected into the tail vein of a mouse.

In one embodiment of the present invention, the saliva germ cell line may be injected into the abdominal cavity of a mouse.

In one embodiment of the present invention, the peripheral blood mononuclear cell and saliva cell line may be injected at a ratio of 5: 3 to 5: 4 for each cell number.

In one embodiment of the present invention, the peripheral blood mononuclear cells may be injected with 0.5 to 5 × 10 ⁶ cells and the salivary gland cell line with 4 to 20 × 10 ⁵ cells.

In one embodiment of the present invention, the injection of peripheral blood mononuclear cells and salivary gland cell line may be performed after 6 to 8 days after transplanting the salivary gland tissue into a mouse.

In addition, the present invention provides a mouse model of Sjogren's syndrome produced by the above method.

The present invention relates to an animal model transplanted with a salivary gland tissue of a patient, wherein an animal model according to the present invention can be provided to provide a patient-customized animal model, while peripheral blood mononuclear cells and salivary cell lines are additionally injected , It is expected that the animal model of Sjogren's syndrome can be produced more effectively, thereby contributing greatly to the development of Sjogren's syndrome treatment and treatment methods.

Figure 1 is a schematic representation of the animal modeling process for pre-clinical human Sjogren's syndrome.
2 is a schematic drawing of a mouse incision surface for salivary gland tissue transplantation.
FIG. 3 is a result of confirming that salivary gland tissue of a human Sjogren's syndrome patient transplanted into a mouse is engrafted and blood vessels are formed.
FIG. 4 shows the results of immunohistochemical staining of mouse and human blood vessels in salivary gland tissues of human Sjogren's syndrome patients and mouse tissues transplanted with PBMCs and saliva cell lines.
FIG. 5 shows the expression of IL-8, MMP3, and IP-10, which are inflammation markers, in histology and immunofluorescence staining of mouse salivary gland tissue and PBMC and saliva cell line in human Sjogren's syndrome patients.
FIG. 6 shows results of flow cytometry comparing human lymphocyte expression patterns in mouse blood when salivary gland tissue of a human Sjogren's syndrome patient is transplanted alone or PBMC and saliva cell lines are sequentially injected after salivary gland transplantation in a human Sjogren's syndrome patient.
FIG. 7 is a graph comparing the expression patterns of human CD4-positive or human CD8-positive T cells in mouse blood when saline tissue of a human Sjogren's syndrome patient is transplanted alone or PBMC and saliva cell lines are sequentially injected after salivary gland transplantation in a human Sjogren's syndrome patient One flow cytometry analysis.
FIG. 8 shows the expression pattern of human CD19-positive B cells or human CD14-positive monocytes in mouse blood when salivary gland tissues of a human Sjogren's syndrome patient were transplanted alone or PBMC and saliva cell lines were sequentially injected after salivary gland transplantation in a human Sjogren's syndrome patient The flow cytometry results are compared.
FIG. 9 is a flow cytometric analysis comparing the expression pattern of human lymphocytes in mouse spleen when salivary gland tissue of a human Sjogren's syndrome patient is transplanted alone or PBMC and saliva cell line are sequentially injected after salivary gland transplantation in a human Sjogren's syndrome patient.
FIG. 10 is a graph comparing the expression patterns of human CD4-positive or human CD8-positive T cells in mouse spleen when PBMCs and saliva cells were sequentially injected after transplantation of salivary gland tissues in patients with human Sjogren's syndrome or saline tissue transplantation in human Sjogren's syndrome patients One flow cytometry analysis.

In studying animal models for studying Sjogren's syndrome, the present invention demonstrates that an animal model of patient-tailored Sjogren's syndrome can be produced effectively when transplanted salivary glands are transplanted into mice and further infused with peripheral blood mononuclear cells and saliva cell lines It was completed by the first confirmation.

Accordingly, the present invention provides a novel animal model of Sjogren's syndrome mouse and a method for producing the same, and the method for producing the animal model of Sjogren's syndrome provided by the present invention is characterized in that the salivary gland tissue isolated from a patient suffering from Sjogren's syndrome is treated with mouse To the patient.

In one embodiment of the present invention, the salivary gland tissue is isolated from patients with Sjogren's syndrome. The salivary glands are washed with PBS, transferred to DMEM medium containing 10% FBS, and then immersed in a mixture of 90% FBS and 10% DMSO They can be stored at -70 ° C until they are transplanted into animal models.

In one embodiment of the present invention, the salivary glands stored at -70 ° C were immersed in a 37 ° C warm bath incubator for transplantation into an animal model, and the melted tissues were washed in DMEM containing 10% FBS, It can be wrapped with matrigel containing human vascular endothelial growth factor (VEGF), and then the tissue can be coated with matrigel at room temperature. In this case, the matrigel is coated using 100 to 1000 쨉 l per separated tissue, preferably 300 쨉 l, and the human VEGF contained therein may be 1 to 500 ng / ml, 10 ng / ml.

In an embodiment of the present invention, an NSG mouse is anesthetized with isofluane, the upper right part of the mouse is epilated, the disinfection alcohol is sprayed, and the 2 to 4 cm of the right hindquarter of the epilated mouse, Preferably, a 3 cm point is cut by 0.5 to 2 cm, preferably 1 cm, with scissors, scissors are inserted into the incised portion, and scissors are opened by 2 to 4 cm, preferably 3 cm above the incision to secure the subcutaneous space. In this case, care should be taken not to tear the film. The end of the incision can then be held with a forceps, the prepared tissue can be plunged into a sterile syringe and placed in the subcutaneous space, and the incision site can be sutured 10 times using a nylon suture. Then, the suture area is disinfected with a disinfectant such as povidone, and the infant is irradiated to look for the consciousness of the mouse. Then, the mouse can be put into a cage and fed with an appropriate amount of feed, and then raised in the breeding room.

In one embodiment of the present invention, peripheral blood mononuclear cells and saliva cell lines isolated from the blood of a patient can be further injected. The peripheral blood mononuclear cell and saliva cell lines are cultured in a ratio of 5: 3 to 5: 4 The peripheral blood mononuclear cells may be injected with 0.5 to 5 × 10 ⁶ cells and the salivary cell line with 4 to 20 × 10 ⁵ cells. Preferably, the peripheral blood mononuclear cells are injected with 1 × 10 ⁶ cells, and the salivary gland cell line can inject 8 × 10 ⁵ cells. In this case, it is preferable that the peripheral blood mononuclear cells are injected into the tail vein and the salivary gland cell line is intraperitoneally injected. The peripheral blood mononuclear cells and the salivary gland cell line are preferably injected after 6 to 8 days from transplanting the salivary gland tissue into the mouse.

The animal model of Sjogren's syndrome as described above was observed three times a week and three times a week after tissue transplantation. Euthanasia was observed 14 days after the transplantation to observe the engraftment and angiogenesis of the salivary tissue. The expression of lymphocytes and human T cells in blood and spleen was confirmed, and successful patient-specific Sjogren's syndrome animal models were constructed by identifying mouse inflammation markers in transplanted human salivary glands.

Meanwhile, the present inventors have found that the patient-tailored Sjögren's syndrome animal model is more successfully produced when the patient's peripheral blood mononuclear cells and saliva cell lines are injected after transplantation of the salivary gland tissue, compared with transplanting the salivary gland tissue alone.

The term " patient-customized animal model ", also referred to herein as an "avatar mouse ", refers to a xenograft animal model produced by stereotactic xenotransplantation of a patient-derived cell or tissue into an immunodeficient animal, A condition in which the disease and the morphological environment of the patient are the same or similar, the genetic environment is the same or similar, the expression characteristics of the marker protein of the disease are the same, and the genetic, physiological and environmental characteristics Can be provided.

The term "immunodeficient animal " of the present invention means an animal model prepared by artificially damaging some components constituting the immune system at the genetic level so that the cancer can be developed, so that a normal immune system is not implemented. The immunodeficient animal may be an animal in which a nervous system has been formed. Preferably, an immunodeficient mammal may be used. More preferably, the immunodeficient animal is an immunodeficient rodent such as a mouse, rat, hamster, guinea pig, (NOD scid gamma) mouse, a non-obese diabetic mouse, a severe combined immunodeficiency (SCID) mouse, a NOD-SCID mouse, a NOG (NOD / SCID Il2rg- / -) mouse, and the like, but it is not particularly limited thereto.

The term " xenotransplantation " of the present invention means a method of transplanting organs, organs, tissues, cells, etc., such as liver, heart, and kidney of another animal. For the purpose of the present invention, the xenotransplantation can be understood as a method of transplanting salivary gland tissue or salivary gland cells isolated from a patient into an immunodeficient animal, but is not particularly limited thereto.

The term "tailored medicine" of the present invention is also referred to as an order-made medicine or a personalized medicine, and the constitution or the environment of a patient is individually examined to determine a suitable treatment method Or a method of treatment.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

< Example > Sjogren's syndrome Preclinical  How to make an animal model

1. Preparation of salivary gland tissue

The salivary gland tissue isolated from patients with Sjogren's syndrome is washed with PBS, transferred to DMEM medium containing 10% FBS, and the separated tissues are immersed in 90% FBS and 10% DMSO solution and stored at -70 ° C.

2. Prepare a tissue implant

Immerse the salivary gland tissue stored at -70 ° C in a 37 ° C water bath and wash it in DMEM medium containing 10% FBS. Human VEGF was added to Matrigel (300 쨉 l) at a concentration of 10 ng / ml, the prepared salivary gland tissue was wrapped with the matrigel, and the Matrigel was hardened.

3. Transplantation of salivary glands

An NSG mouse is anesthetized with respiration using isoflurane, and the upper part of the right hindquarter of the mouse is epilated. After disinfection alcohol is sprayed on the right buttocks of the depilated mouse, the upper 3cm area is cut by 1cm with scissors, and the scissors are inserted into the cut part to open the scissors 3cm above the upper part of the lid to secure the subcutaneous space Reference). The end of the incised portion is held with a forceps, and the tissue wrapped with the matrigel is drained with a sterilized spoon and put in the secured subcutaneous space, and the incision site is sutured about 10 times using a nylon suture. After the suture, sterilize the surgical site with povidone, apply infrared light, etc., and check that the mouse finds consciousness and then raise it in the breeding room.

4. Invasion of salivary gland cell line

Seven days after transplantation, 1.5 × 10 ⁶ PBMCs (Peripheral Blood Mononuclear Cell) isolated from the blood of patients with Sjogren's Syndrome were suspended in 100 μl of PBS and injected into the tail vein of the mouse. At the same time, -253 cells (8 x 10 ⁵ ) are suspended in 100 μl of PBS and administered intraperitoneally to mice.

< Experimental Example >

1. Identification of engraftment and angiogenesis of salivary glands in an animal model of Sjogren's syndrome

NSG mice prepared by the above example were euthanized 14 days after transplantation of salivary gland tissue, and the graft site was excised to confirm the engraftment and vascularization of the salivary gland tissue. As a result, as can be seen from FIG. 3, the transplanted salivary gland tissues were properly adhered to the mouse, and new blood vessels were visually confirmed to be formed.

2. Human in Sjogren's Syndrome Animal Model Vascular cell  Confirm

To confirm whether an animal model of human Sjogren's syndrome was produced properly, the mouse produced by the above example was euthanized 14 days after transplantation of salivary gland tissue and human angiogenesis was observed by immunofluorescent staining of angiogenic marker CD31 .

To this end, the frozen sections of transplanted salivary glands were placed in OCT compound, cut into a 7 μm thick slice on a gelatin-coated slide glass, fixed at room temperature with 4% paraformaldehyde buffer, After washing with phosphate buffer, serum of normal chlorine diluted 10-fold with phosphate buffer was treated at room temperature for 1 hour to inhibit nonspecific antibody binding of salivary gland tissue. Next, the anti-human CD31 (PECAM-1) Phycoerythrin (PE) antibody, which indicates that it is a human vascular cell, was used to express anti-mouse CD31 (PECAM-1) fluorescein isothiocyanate (FITC) After incubation at room temperature, the cells were washed with phosphate buffer solution for 15 minutes to remove residual antibody. Then, they were sealed with a sealing agent containing DAPI, which is a nuclear fluorescent dye, and then dried and observed with a confocal microscope.

As a result, as shown in Fig. 4, human vascular cells were confirmed in the mouse transplanted with the salivary gland tissue of the present invention and the PBMC and saliva cell line, and it was found that a mouse model in which a human salivary gland tissue was transplanted was well formed.

3. Histochemical staining and Immunofluorescent staining  Inflammation through Marker  Confirm

The present inventors observed that inflammation markers were observed by immunohistochemistry and immunofluorescence staining by euthanizing the mouse produced through the above example 14 days after transplantation of salivary gland tissue and taking salivary gland tissue sections.

Immunohistochemistry was performed by firstly immersing the salivary glands of euthanized mouse animal models in 10% neutral formalin, formatting them in paraffin, and cutting them into 7 μm sections. The sections were stained with H & E staining and observed with an optical microscope. Immunofluorescence staining was performed according to the method described in Experimental Example 2 above.

As a result, as shown in FIG. 5, IL-8, MMP3, and IP-10, which are inflammation markers, were expressed in tissues transplanted with salivary gland tissue.

4. Flow cell  Through analysis Sogren  Identifying Syndrome Animal Models

The present inventors confirmed that an animal model of Sjogren's syndrome can be produced by transplanting salivary glands of a human Sjögren's syndrome in an NSG mouse in the initial experiment. While various experiments were conducted to improve the production effect of Sjören's syndrome animal model, Injection of PBMC and salivary gland cells in patients with Sjögren's syndrome showed that the effect of Sjögren's syndrome animal model was improved.

This was confirmed by flow cytometry. In the above example, it was confirmed that the mouse or saline tissue transplanted in the Sjogren's Syndrome patient was transferred to the blood or saliva from the mouse in which the PBMC and the saliva cell line were further infused with Sjogren's Syndrome After the spleen was collected, the cells were reacted with human lymphocytes, CD4 positive T cells, CD8 positive T cells, CD 19 positive B cells and CD14 positive mononuclear specific antibodies for 30 minutes, followed by washing with phosphate buffer solution, And analyzed by flow cytometry.

As a result, as shown in FIG. 6 to FIG. 10, it was found that, in comparison with mice transplanted with salivary gland tissue in both human lymphocytes, CD4 + T cells, CD8 + T cells, CD19 + B cells and CD14 + Day, it was confirmed that the expression was much higher in mice injected with PBMC and saliva.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (11)

A method of producing an animal model of Sjogren's syndrome comprising implanting salivary gland tissue isolated from a Sjogren's syndrome patient into a mouse. The method according to claim 1,
Wherein the salivary gland tissue has a volume of 0.1 cm 3 to 2 cm 3. The method according to claim 1,
Wherein the salivary gland tissue is coated with Matrigel and transplanted. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; The method according to claim 1,
Wherein said salivary gland tissue is transplanted into a subcutaneous space secured at an upper portion of 2 to 4 cm from an upper part of said salivary gland with a distance of 2 to 4 cm from said upper part of said guttural tissue, . The method according to claim 1,
Wherein the method comprises transplanting salivary gland tissue isolated from a patient with Sjogren's syndrome into a mouse and then injecting further peripheral blood mononuclear cells and saliva cell lines separated from the Sjogren's syndrome patient. 6. The method of claim 5,
Wherein said peripheral blood mononuclear cells are injected into the tail vein of a mouse. 6. The method of claim 5,
Wherein the saliva gland cell line is injected into the abdominal cavity of a mouse. 6. The method of claim 5,
Wherein said peripheral blood mononuclear cell and saliva cell line are injected so that the ratio of the number of cells to the number of cells is 5: 3 to 5: 4. 6. The method of claim 5,
Wherein said peripheral blood mononuclear cells are injected with 0.5 to 5 x 10 &lt; ⁶ &gt; cells and said salivary gland cells with 4 to 20 x 10 ⁵ cells are injected. 6. The method of claim 5,
Wherein said peripheral blood mononuclear cells and salivary gland cells are injected after implantation of said salivary gland tissue in a mouse for 6 to 8 days. A Sjören syndrome mouse animal model prepared by the method of claim 1.

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