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WO2019190216A1 - Composition pour améliorer l'infiltration dans des tissus biologiques comprenant du triton x-100 et de l'urée en tant que principes actifs, et procédé d'amélioration de l'infiltration dans des tissus biologiques l'utilisant - Google Patents

Composition pour améliorer l'infiltration dans des tissus biologiques comprenant du triton x-100 et de l'urée en tant que principes actifs, et procédé d'amélioration de l'infiltration dans des tissus biologiques l'utilisant Download PDF

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Publication number
WO2019190216A1
WO2019190216A1 PCT/KR2019/003621 KR2019003621W WO2019190216A1 WO 2019190216 A1 WO2019190216 A1 WO 2019190216A1 KR 2019003621 W KR2019003621 W KR 2019003621W WO 2019190216 A1 WO2019190216 A1 WO 2019190216A1
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WO
WIPO (PCT)
Prior art keywords
urea
triton
composition
biological tissues
enhancing
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/KR2019/003621
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English (en)
Korean (ko)
Inventor
박영일
금상일
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Binaree Co Ltd
Original Assignee
Binaree Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020180174047A external-priority patent/KR20190114728A/ko
Application filed by Binaree Co Ltd filed Critical Binaree Co Ltd
Publication of WO2019190216A1 publication Critical patent/WO2019190216A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers

Definitions

  • the present invention relates to a composition for enhancing biological tissue penetration, comprising Triton X-100 and urea as active ingredients, and a method for enhancing tissue penetration using the same.
  • the microstructure of the biopsy or the like is fixed and embedded in paraffin to make a nanometer-sized slide, and the microstructure is analyzed by optical or fluorescence.
  • a microscope such as a confocal is used, and in this case, information of tens of micrometers in thickness can be obtained. But all of this is limited by the depth through which the light source can penetrate. Therefore, in order to obtain a three-dimensional image of thicker tissue, it is necessary to continuously slice the tissue, image it with a microscope, and then reconstruct it.
  • Tissue clearing technology can confirm the structure and protein expression without damaging the tissue, so recently, a technique for transparent tissue has been developed in a variety of ways.
  • Existing tissue clearing techniques have been reported for antigen preservation of tissues treated by the Spatleholz, BABB, Scale S, iDISCO method of tissue clearing method using an organic solvent, and active CLARITY technology (ACT) method of polymer injection. Except for ACT, there is a problem in that fluorescence and antigen retention are reduced. ACT has more than 90% antigen retention, which shows higher retention compared to methods requiring binding to hydrogel polymers in addition to immobilized proteins such as CLARITY.
  • ACT active CLARITY technology
  • immunostaining refers to a staining method using an antibody developed by a method of studying the localization of a specific protein in cells or tissues.
  • a secondary antibody in which fluorescent substances such as fluorescent dyes are added to the primary antibody is acted on and observed with an optical microscope. The information that is usually obtained because of fixed observation of cells or tissues is static, but the use of secondary antibodies with different fluorescence colors makes it possible to study colocalization of plural proteins. It is becoming.
  • enzyme immunohistochemical staining is a very widely used method for the origin of various tumors, tumor classification, prognosis, and the like.
  • the need for quantitative immunohistochemical staining has emerged as sheep are known to be important in determining the prognosis of cancer and responsiveness to anticancer agents.
  • immunohistochemical staining method involves reacting and attaching a primary antibody to an antigen, followed by attaching a secondary antibody directly attached to an enzyme and reacting with the substrate solution of the enzyme for color development, or reacting a secondary antibody with biotin. Reacting the reagent with the enzyme to avidin, which has a strong adhesion to biotin (ABC method), and then reacting the substrate solution with this enzyme to develop color and observe the presence of the original antigen under a microscope. Doing.
  • the present inventors make the biological tissue transparent, and infiltrate the biological tissue by infiltrating a polymer material such as an antibody or a dye (dye) into the transparent biological tissue, the antibody or dye (dye) penetrates deeper tissue deeper dyeing
  • a polymer material such as an antibody or a dye (dye) into the transparent biological tissue
  • the antibody or dye (dye) penetrates deeper tissue deeper dyeing
  • the present inventors have found that staining is enhanced by enhancing penetration compared to conventional methods when Triton X-100 and urea are used together when infiltrating a polymer material such as an antibody or dye (dye) into a biological tissue when clearing biological tissue staining. It was confirmed that this invention was completed based on this.
  • compositions for enhancing biological tissue penetration including Triton X-100 and urea as active ingredients.
  • Another object of the present invention is to provide a method for enhancing biological tissue penetration, comprising the step of reacting the composition for enhancing biological tissue penetration with the antibody or dye (dye) to the cleared biological tissue.
  • the present invention provides a composition for enhancing biological tissue penetration comprising Triton X-100 (Triton X-100) and urea (urea) as an active ingredient.
  • the present invention provides a method for enhancing biological tissue penetration, comprising the step of reacting the composition for enhancing biological tissue penetration with the antibody or dye (dye) to the cleared biological tissue.
  • the present invention provides a use for enhancing tissue penetration of a composition comprising Triton X-100 and urea as active ingredients.
  • the concentration of Triton X-100 may be 0.01 to 0.5 v / v%.
  • the concentration of urea may be 0.1 to 5M.
  • the composition may increase the penetration of the antibody or dye (dye).
  • the dye may be a blue dextran.
  • Triton X-100 and urea according to the present invention when used in combination with a tissue for enhancing tissue penetration enhancer composition as an active ingredient has an effect of enhancing the penetration of antibodies or dyes (dye) compared to when not used together
  • the present invention is expected to be useful for identifying causes of various diseases and finding treatments.
  • 1 is a view showing the result of staining the cleared biological tissue using blue dextran using 0.1v / v% Triton X-100 and 1M urea according to an embodiment of the present invention.
  • Figure 2 compares the results of staining the cleared biological tissue with blue dextran using 0.1v / v% Triton X-100 and 1M urea in accordance with an embodiment of the present invention when using water alone The figure shown.
  • the present invention provides a composition for enhancing biological tissue penetration, including Triton X-100 and urea as active ingredients.
  • the present invention provides a method for enhancing biological tissue penetration, comprising the step of reacting the composition for enhancing biological tissue penetration with the antibody or dye (dye) to the cleared biological tissue.
  • Triton X-100 of the present invention is a nonionic surfactant having a hydrophilic polyethylene oxide group and a hydrocarbon lipophilic or hydrophobic group, and is added as a solubilizer when separating and refining its components in cells, tissues, and cell membranes.
  • radioactivity e.g., 3H
  • radioactive intercalation in tissue cells or aqueous fractions is added to the sample as a solvent when the liquid scintillator is used, so that it can be easily mixed in the scintillator solution. have.
  • the urea (urea) of the present invention is an organic compound having the chemical formula CO (NH 2 ) 2 , and has no color or odor, and forms a columnar crystal, having a molecular weight of 60.047 and a melting point of 132.7 ° C. (1 atm). , Specific gravity is 1.335. It is a highly polar substance, so it is well soluble in water and alcohol but insoluble in ether, and is used for separating fertilizers, raw materials of urea resins, diuretics, analytical reagents, and organic compounds.
  • the concentration of the Triton X-100 may be 0.01 to 0.5v / v%, according to one embodiment of the present invention 0.01 to 0.4v / v%, 0.01 to 0.3v / v%, 0.01 to 0.2v / v%, or 0.1v / v%, but is not limited thereto.
  • the concentration of the urea may be 0.1 to 5M, according to one embodiment of the present invention may be 0.1 to 4M, 0.1 to 3M, 0.1 to 2M, or 1M, but is not limited thereto.
  • the composition may increase penetration of an antibody or dye, but is not limited thereto.
  • the dye may be a blue dextran, but is not limited thereto.
  • the blue dextran is a combination of Cibacron blue F3GA to dextran having an average molecular weight of 2 million, so that the solutes that cannot enter the mesh of the support gel in gel filtration such as proteins are pores between the gel particles. Elution is carried out in the elution volume (called void volume or exclusion volume) corresponding to A blue solution of blue dextran is used to determine this volume.
  • Example 1 Penetration enhancing effect of dye dye into biological tissue using composition for enhancing tissue penetration
  • the composition for enhancing biological tissue penetration of the present invention when using 0.1v / v% Triton X-100 and 1M urea, the composition for enhancing biological tissue penetration of the present invention at the time of dyeing the biological tissues, it is confirmed that the coloration is more apparent by increasing the penetration of dyes. It was.
  • composition for enhancing tissue penetration of Triton X-100 and urea can observe structural images of biological tissues in detail and analyze changes in tissues that are difficult to observe, causing various diseases. It is expected to be usefully used to identify and to find treatments.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne une composition pour améliorer l'infiltration dans des tissus biologiques, comprenant du Triton X-100 et de l'urée en tant que principes actifs, et un procédé d'amélioration de l'infiltration dans des tissus biologiques l'utilisant. La composition pour améliorer l'infiltration dans des tissus biologiques, comprenant du Triton X-100 et de l'urée en tant que principes actifs selon la présente invention, lorsqu'elle est utilisée en association pour colorer des tissus biologiques, sert à améliorer l'infiltration d'anticorps ou d'un colorant, par rapport au cas où elle n'est pas utilisée, et permet ainsi d'examiner l'image structurale des tissus biologiques plus en détail et permet des analyses sur des tissus difficiles à examiner. Ainsi, la présente invention peut être avantageusement utilisée pour identifier les causes de diverses maladies et découvrir des procédés de traitement associés.
PCT/KR2019/003621 2018-03-29 2019-03-28 Composition pour améliorer l'infiltration dans des tissus biologiques comprenant du triton x-100 et de l'urée en tant que principes actifs, et procédé d'amélioration de l'infiltration dans des tissus biologiques l'utilisant Ceased WO2019190216A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR20180036814 2018-03-29
KR10-2018-0036814 2018-03-29
KR10-2018-0174047 2018-12-31
KR1020180174047A KR20190114728A (ko) 2018-03-29 2018-12-31 트리톤 x-100 및 우레아를 유효성분으로 포함하는 생체조직 침투 증강용 조성물 및 이를 이용한 생체조직 침투 증강 방법

Publications (1)

Publication Number Publication Date
WO2019190216A1 true WO2019190216A1 (fr) 2019-10-03

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PCT/KR2019/003621 Ceased WO2019190216A1 (fr) 2018-03-29 2019-03-28 Composition pour améliorer l'infiltration dans des tissus biologiques comprenant du triton x-100 et de l'urée en tant que principes actifs, et procédé d'amélioration de l'infiltration dans des tissus biologiques l'utilisant

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024177108A1 (fr) * 2023-02-22 2024-08-29 国立大学法人大阪大学 Polymère transparent permettant l'observation en profondeur d'un tissu tridimensionnel vivant

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010104441A (ko) * 2000-04-28 2001-11-26 송우근 씨형 간염바이러스의 코아 항원 단백질 및 그 친수적분절의 발현 및 정제 방법
US6472216B1 (en) * 2001-07-24 2002-10-29 Ann-Shyn Chiang Aqueous tissue clearing solution
US20140178927A1 (en) * 2011-05-20 2014-06-26 Riken Clarifying reagent for biological materials and use thereof
US20150168417A1 (en) * 2013-12-12 2015-06-18 University Of Houston System Universal Antigen Retrieval Compounds and Methods of Use
US20160266016A1 (en) * 2013-08-14 2016-09-15 Riken Composition for preparing biomaterial with excellent light-transmitting property, and use thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010104441A (ko) * 2000-04-28 2001-11-26 송우근 씨형 간염바이러스의 코아 항원 단백질 및 그 친수적분절의 발현 및 정제 방법
US6472216B1 (en) * 2001-07-24 2002-10-29 Ann-Shyn Chiang Aqueous tissue clearing solution
US20140178927A1 (en) * 2011-05-20 2014-06-26 Riken Clarifying reagent for biological materials and use thereof
US20160266016A1 (en) * 2013-08-14 2016-09-15 Riken Composition for preparing biomaterial with excellent light-transmitting property, and use thereof
US20150168417A1 (en) * 2013-12-12 2015-06-18 University Of Houston System Universal Antigen Retrieval Compounds and Methods of Use

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024177108A1 (fr) * 2023-02-22 2024-08-29 国立大学法人大阪大学 Polymère transparent permettant l'observation en profondeur d'un tissu tridimensionnel vivant

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