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 PDFInfo
- 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
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- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 239000004202 carbamide Substances 0.000 title claims abstract description 29
- 230000002708 enhancing effect Effects 0.000 title claims abstract description 29
- 239000000203 mixture Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 25
- 239000004480 active ingredient Substances 0.000 title claims abstract description 10
- 230000008595 infiltration Effects 0.000 title abstract 6
- 238000001764 infiltration Methods 0.000 title abstract 6
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 title 1
- 229920004890 Triton X-100 Polymers 0.000 claims abstract description 26
- 239000013504 Triton X-100 Substances 0.000 claims abstract description 26
- 239000000975 dye Substances 0.000 claims description 30
- 230000035515 penetration Effects 0.000 claims description 29
- 229920002307 Dextran Polymers 0.000 claims description 11
- 239000003961 penetration enhancing agent Substances 0.000 claims description 5
- 238000010186 staining Methods 0.000 abstract description 10
- 201000010099 disease Diseases 0.000 abstract description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 4
- 238000011282 treatment Methods 0.000 abstract description 3
- 238000004458 analytical method Methods 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 7
- 239000000427 antigen Substances 0.000 description 6
- 108091007433 antigens Proteins 0.000 description 6
- 102000036639 antigens Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- QURLONWWPWCPIC-UHFFFAOYSA-N 2-(2-aminoethoxy)ethanol;3,6-dichloro-2-methoxybenzoic acid Chemical compound NCCOCCO.COC1=C(Cl)C=CC(Cl)=C1C(O)=O QURLONWWPWCPIC-UHFFFAOYSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000012744 immunostaining Methods 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 150000002894 organic compounds Chemical class 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 239000002861 polymer material Substances 0.000 description 2
- 238000004393 prognosis Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 108090001008 Avidin Proteins 0.000 description 1
- 239000004215 Carbon black (E152) Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010058683 Immobilized Proteins Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229920001807 Urea-formaldehyde Polymers 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000012223 aqueous fraction Substances 0.000 description 1
- 238000001574 biopsy Methods 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- YKCWQPZFAFZLBI-UHFFFAOYSA-N cibacron blue Chemical compound C1=2C(=O)C3=CC=CC=C3C(=O)C=2C(N)=C(S(O)(=O)=O)C=C1NC(C=C1S(O)(=O)=O)=CC=C1NC(N=1)=NC(Cl)=NC=1NC1=CC=CC=C1S(O)(=O)=O YKCWQPZFAFZLBI-UHFFFAOYSA-N 0.000 description 1
- 230000008045 co-localization Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000002934 diuretic Substances 0.000 description 1
- 229940030606 diuretics Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000007863 gel particle Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 229930195733 hydrocarbon Natural products 0.000 description 1
- 150000002430 hydrocarbons Chemical class 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- 229920013746 hydrophilic polyethylene oxide Polymers 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000009830 intercalation Methods 0.000 description 1
- 230000002687 intercalation Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
Images
Classifications
-
- 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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- 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.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR10-2018-0036814 | 2018-03-29 | ||
| KR20180036814 | 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 |
Family
ID=68060296
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/KR2019/003621 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 |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2019190216A1 (fr) |
Citations (5)
| 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 |
-
2019
- 2019-03-28 WO PCT/KR2019/003621 patent/WO2019190216A1/fr active Application Filing
Patent Citations (5)
| 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 |
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