WO2019190216A1 - Composition for enhancing infiltration into biological tissues comprising triton x-100 and urea as active ingredients, and method of enhancing infiltration into biological tissues using same - Google Patents

Composition for enhancing infiltration into biological tissues comprising triton x-100 and urea as active ingredients, and method of enhancing infiltration into biological tissues using same Download PDF

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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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urea
triton
composition
biological tissues
enhancing
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박영일
금상일
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주식회사 바이나리
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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

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  • 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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Abstract

The present invention relates to a composition for enhancing infiltration into biological tissues, comprising Triton X-100 and urea as active ingredients, and a method of enhancing infiltration into biological tissues using same. The composition for enhancing infiltration into biological tissues, comprising Triton X-100 and urea as active ingredients according to the present invention, when used together for staining biological tissues, serves to enhance the infiltration of antibodies or a dye, compared to when it is not used, and thus allows the structural image of the biological tissues to be examined in greater detail and enables analyses on tissues that are difficult to examine. Thus, the present invention may be beneficially used in identifying the causes of various diseases and discovering treatment methods therefor.

Description

트리톤 X-100 및 우레아를 유효성분으로 포함하는 생체조직 침투 증강용 조성물 및 이를 이용한 생체조직 침투 증강 방법 Composition for enhancing tissue penetration by Triton X-100 and urea as an active ingredient and method for enhancing tissue penetration using same
본 발명은 트리톤 X-100(Triton X-100) 및 우레아(urea)를 유효성분으로 포함하는 생체조직 침투 증강용 조성물 및 이를 이용한 생체조직 침투 증강 방법에 관한 것이다.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.
현재 질병의 진단은 2차원 스캐닝을 통한 3차원으로의 재구성 기술에 의해 보다 정교하게 진단할 수 있는 기술로 발전하여 왔다. 그러나, 현재 개발된 기술은 밀리미터 수준으로 세포 수준에서의 분석이 가능한 정도의 기술은 매우 미흡한 실정이다.Currently, the diagnosis of disease has been developed into a technology that can be more precisely diagnosed by the reconstruction technology in three dimensions through two-dimensional scanning. However, currently developed technology is a millimeter level, the technology that can be analyzed at the cellular level is very insufficient.
즉, 생검 등의 생체조직을 고정한 후 파라핀 등으로 포매하여 나노미터 두께의 슬라이드를 만들어 광학이나 형광 등으로 미세 구조를 분석한다. 이러한 미세 구조에 대한 이미지 기술을 이용하여 3차원 이미지를 구성하기 위해서는 콘포칼과 같은 현미경을 이용해야 하며 이러한 경우 수십 마이크로 미터 수준 두께의 정보를 획득할 수 있다. 그러나 이 모든 것은 광원이 침투 할 수 있는 깊이에 의해 제한된다. 따라서, 보다 두꺼운 조직의 3차원 이미지를 획득하기 위해서는 조직을 연속적으로 절편하여 현미경으로 이미징 한 후 다시 재구성하는 과정이 필요하다.In other words, 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. In order to construct a three-dimensional image by using an image technology of such a microstructure, 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.
조직 투명화 기술은 조직의 손상 없이 구조 및 단백질 발현 등을 확인 할 수 있으므로 최근에 매우 다양한 방법으로 조직을 투명화 할 수 있는 기술이 개발되었다. 기존의 조직 투명화 기술은 유기용매를 이용한 조직 투명화 방법인 Spatleholz, BABB, Scale S, iDISCO법과, 폴리머 주입법인 ACT(active CLARITY technology)법에 의해 처리된 조직의 항원 보존성이 보고된 바 있다. ACT를 제외한 다른 방법의 경우 형광과 항원의 보존성이 감소하는 문제를 가지고 있다. ACT의 경우 90% 이상의 항원 보존성을 가지며, 이는 클라리티(CLARITY)와 같이 고정된 단백질에 추가로 하이드로젤 폴리머와의 결합을 필요로 하는 방법에 비하면 보다 높은 보존성을 보인다. 그러나 강한 조직 고정 과정은 항원성의 손실을 유발하여, 사용할 수 있는 항체가 감소하는 등의 문제점을 고려해야 하므로, 여러 가지 기술의 개선이 필요하다.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. However, a strong tissue fixation process causes a loss of antigenicity, which requires consideration of problems such as a decrease in the number of available antibodies.
한편, 면역염색은 세포 또는 조직 등에서 특정한 단백질의 국재를 연구하는 방법으로 개발한 항체를 사용하는 염색법을 말한다. 1차항체에 형광색소 등의 형광물질을 부가한 2차항체를 작용시켜 광학현미경으로 관찰한다. 세포 또는 조직을 고정하여 관찰하기 때문에 통상 얻게 되는 정보는 정적인 것이지만 형광색이 다른 2차항체를 사용하면, 복수인 단백질의 공동국재도 연구할 수 있는 점에서 분자세포생물학에 있어서도 매우 중요한 기술로 인식되고 있다.On the other hand, 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.
또한, 효소 면역조직화학 염색법은 각종 종양의 기원, 종양의 분류, 예후판정 등의 목적으로 매우 광범위하게 이용되고 있는 방법으로, 특히 최근에는 어떤 항원의 존재 여부뿐 아니라 조직이나 세포에 존재하는 항원의 양이 암의 예후나 항암제에 대한 반응성을 결정짓는데 중요하다는 사실이 알려지면서 정량적인 면역조직화학 염색의 필요성이 대두되었다.In addition, 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.
그러나, 현재 이용 가능한 면역조직화학 염색법은 항원에 대한 일차 항체를 반응시켜 붙인 다음 직접 효소가 붙은 이차 항체를 붙이고 효소의 기질액을 반응시켜 발색을 시키거나, 바이오틴이 붙은 이차항체를 반응 시키고 이후에 바이오틴과 강력하게 붙는 성질이 있는 아비딘에 효소가 달라붙어있는 시약을 반응 시키고(ABC 법) 이후에 이 효소에 대한 기질액을 반응시켜서 색깔을 발색시켜 현미경하에서 원래 항원의 존재를 관찰하는 방법을 사용하고 있다.However, currently available 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.
따라서, 상기와 같은 면역염색법에서는 항원에 대한 항체를 반응시켜 선명한 발색을 얻는 것이 중요하다. Therefore, in the above immunostaining method, it is important to obtain a clear color by reacting the antibody against the antigen.
이에, 본 발명자들은 생체조직을 투명화 시키고, 투명화된 생체조직에 항체 또는 염료(dye)와 같은 고분자 물질을 침투시켜 생체조직을 염색할 경우 상기 항체 또는 염료(dye)를 보다 조직 깊이 침투시켜 염색을 선명하게 시킬 수 있는 조성물 및 상기 조성물을 이용한 생체조직 침투 증강 방법을 개발하였다.Therefore, 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 composition capable of sharpening and a method of enhancing tissue penetration using the composition have been developed.
본 발명자들은 투명화된 생체조직 염색 시 생체조직에 항체 또는 염료(dye)와 같은 고분자 물질을 침투시킬 때 Triton X-100 및 urea를 함께 사용하는 경우 종래의 방법에 비해 침투를 증강시켜 염색이 더 잘되는 것을 확인하고, 이에 기초하여 본 발명을 완성하였다.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.
이에, 본 발명의 목적은 트리톤 X-100(Triton X-100) 및 우레아(urea)를 유효성분으로 포함하는 생체조직 침투 증강용 조성물을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a composition for enhancing biological tissue penetration, including Triton X-100 and urea as active ingredients.
본 발명의 다른 목적은 투명화된 생체조직에 상기 생체조직 침투 증강용 조성물을 항체 또는 염료(dye)와 함께 반응시키는 단계를 포함하는, 생체조직 침투 증강 방법을 제공하는 것이다.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.
그러나, 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술 분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned can be clearly understood by those skilled in the art from the following description. There will be.
상기와 같은 목적을 달성하기 위하여, 본 발명은 트리톤 X-100(Triton X-100) 및 우레아(urea)를 유효성분으로 포함하는 생체조직 침투 증강용 조성물을 제공한다.In order to achieve the above object, 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.
또한, 본 발명은 투명화된 생체조직에 상기 생체조직 침투 증강용 조성물을 항체 또는 염료(dye)와 함께 반응시키는 단계를 포함하는, 생체조직 침투 증강 방법을 제공한다.In addition, 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.
또한, 본 발명은 트리톤 X-100(Triton X-100) 및 우레아(urea)를 유효성분으로 포함하는 조성물의 생체조직 침투 증강 용도를 제공한다.In addition, the present invention provides a use for enhancing tissue penetration of a composition comprising Triton X-100 and urea as active ingredients.
본 발명의 일구현예로서, 상기 Triton X-100의 농도는 0.01 내지 0.5 v/v%일 수 있다.In one embodiment of the present invention, the concentration of Triton X-100 may be 0.01 to 0.5 v / v%.
본 발명의 다른 구현예로서, 상기 urea의 농도는 0.1 내지 5M일 수 있다.In another embodiment of the present invention, the concentration of urea may be 0.1 to 5M.
본 발명의 또 다른 구현예로서, 상기 조성물은 항체 또는 염료(dye)의 침투를 증가시킬 수 있다.As another embodiment of the present invention, the composition may increase the penetration of the antibody or dye (dye).
본 발명의 또 다른 구현예로서, 상기 염료(dye)는 블루 덱스트란(blue dextran)일 수 있다.In another embodiment of the present invention, the dye may be a blue dextran.
본 발명에 따른 Triton X-100 및 urea를 유효성분으로 포함하는 생체조직 침투 증강용 조성물을 생체 조직 염색 시 함께 사용할 경우 사용하지 않는 경우에 비해 항체 또는 염료(dye)의 침투를 증강시키는 효과가 있어 생체 조직의 구조적 이미지를 보다 자세하게 관찰할 수 있고 관찰하기 어려운 조직의 변화를 분석할 수 있으므로, 본 발명은 다양한 질환의 원인을 규명하고 치료법을 찾아내는 데 유용하게 사용될 수 있을 것으로 기대된다.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 As structural images of biological tissues can be observed in more detail, and tissue changes that are difficult to observe can be analyzed, the present invention is expected to be useful for identifying causes of various diseases and finding treatments.
도 1은 본 발명의 일구현예에 따른 0.1v/v% Triton X-100과 1M urea를 사용하여 투명화된 생체 조직을 blue dextran으로 염색한 결과를 나타낸 도면이다.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.
도 2는 본 발명의 일구현예에 따른 0.1v/v% Triton X-100과 1M urea를 물과 함께 사용하여 투명화된 생체 조직을 blue dextran으로 염색한 결과를 물을 단독으로 사용하였을 때와 비교하여 나타낸 도면이다.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.
본 발명은 트리톤 X-100(Triton X-100) 및 우레아(urea)를 유효성분으로 포함하는 생체조직 침투 증강용 조성물을 제공한다.The present invention provides a composition for enhancing biological tissue penetration, including Triton X-100 and urea as active ingredients.
또한, 본 발명은 투명화된 생체조직에 상기 생체조직 침투 증강용 조성물을 항체 또는 염료(dye)와 함께 반응시키는 단계를 포함하는, 생체조직 침투 증강 방법을 제공한다.In addition, 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.
본 발명의 상기 트리톤 X-100(Triton X-100)은 hydrophilic polyethylene oxide group과 hydrocarbon lipophilic 또는 hydrophobic group을 가지고 있는 비이온성 surfactant 로서, 세포, 조직, 세포막에서 그의 구성성분을 분리 정제할 때에 용해제로 첨가하거나, 조직세포나 수용성 분획에 끼어들어간 방사성동위원소(예: 3H)의 방사능 측정을 액체 신틸레이터를 사용하였을 때 시료의 용제로 첨가하여 섬광기 용액에 혼합되기 쉽도록 하는 등 많은 용도로 사용하고 있다.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. Or radioactivity (e.g., 3H) of 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.
또한, 본 발명의 상기 우레아(요소; urea)는 화학식이 CO(NH 2) 2인 유기화합물로, 색이나 냄새가 없고 기둥 모양의 결정을 만드는 물질이며, 분자량 60.047, 녹는점 132.7℃(1atm), 비중 1.335이다. 극성이 강한 물질이어서 물과 알코올에는 잘 녹지만 에테르에는 녹지 않으며, 비료, 요소 수지의 원료, 이뇨제, 분석용 시약, 및 유기 화합물의 분리 등에 사용되고 있다. In addition, 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.
이 때, 상기 Triton X-100의 농도는 0.01 내지 0.5v/v%일 수 있고, 본 발명의 일 구현예에 따르면 0.01 내지 0.4v/v%, 0.01 내지 0.3v/v%, 0.01 내지 0.2v/v%, 또는 0.1v/v%일 수 있으나, 이에 제한되지 않는다.At this time, 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.
또한, 상기 urea의 농도는 0.1 내지 5M일 수 있고, 본 발명의 일 구현예에 따르면 0.1 내지4M, 0.1 내지 3M, 0.1 내지 2M, 또는 1M일 수 있으나, 이에 제한되지는 않는다.In addition, 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.
본 발명에서 상기 조성물은 항체 또는 염료(dye)의 침투를 증가시킬 수 있으나 이에 제한되지는 않는다.In the present invention, the composition may increase penetration of an antibody or dye, but is not limited thereto.
이 때, 상기 염료(dye)는 블루 덱스트란(blue dextran)일 수 있으나, 이에 제한되지는 않는다.In this case, the dye may be a blue dextran, but is not limited thereto.
상기 블루 덱스트란(blue dextran)은 평균분자량 200만인 덱스트란(dextran)에 시바크론블루 F3GA를 결합시킨 것으로, 단백질 등 겔 여과에 있어 지지체 겔의 망목에는 들어갈 수 없는 정도인 용질은 겔 입자간의 공극의 전용적에 상당하는 용출용적(일명, 보이드(void)용적 또는 배제용적)으로 용출한다. 블루 덱스트란의 푸른 용액은 이 용적을 구하기 위해 사용하고 있다.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.
본 발명의 일실시예에서는 0.1v/v% Triton X-100 및 1M urea를 이용하여 blue dextran으로 생체 조직의 염색을 실시한 결과, 0.1v/v% Triton X-100 및 1M urea를 생체 조직 염색 시 함께 사용할 경우, 사용하지 않은 경우에 비해 염료(dye)의 침투를 증가시켜 발색이 선명하게 나타나는 것을 확인하였다(실시예 1 참조). In one embodiment of the present invention as a result of staining the biological tissue with blue dextran using 0.1v / v% Triton X-100 and 1M urea, when staining 0.1v / v% Triton X-100 and 1M urea When used together, it was confirmed that color development appeared clearly by increasing the penetration of dye (dye) compared with the case without using (see Example 1).
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.
실시예1Example 1 . 생체 조직 침투 증강용 조성물을 이용한 생체 조직으로의 염색 dye의 침투 증강 효과. Penetration enhancing effect of dye dye into biological tissue using composition for enhancing tissue penetration
24주령 mouse whole brain을 121℃, 15 psi로 15분 반응시킨 후 본 발명의 생체 조직 침투 증강용 조성물인 0.1v/v% Triton X-100 및 1M urea를 이용하여 70kD blue dextran을 20mg/ml의 농도로 4℃에서 24시간 동안 반응시켰다.After reacting 24-week-old mouse whole brain at 121 ° C. and 15 psi for 15 minutes, 70 mg of blue dextran was added to 20 mg / ml using 0.1v / v% Triton X-100 and 1M urea, which are compositions for enhancing tissue penetration. The reaction was carried out at 4 ° C. for 24 hours.
생체 조직 염색 시 0.1v/v% Triton X-100 및 1M urea를 단독으로 사용한 경우와 함께 사용한 경우 염색 결과를 도 1에 나타내었다.When stained with a tissue when used with 0.1v / v% Triton X-100 and 1M urea alone is shown in Figure 1 staining results.
또한, 생체 조직 염색 시 0.1v/v% Triton X-100 및 1M urea를 물과 함께 사용하였을 때의 염색 결과를 물만 사용하였을 때와 비교하여 도 2에 나타내었다. In addition, the staining results of using 0.1v / v% Triton X-100 and 1M urea with water when staining biological tissues are shown in FIG. 2 in comparison with water only.
그 결과, 본 발명의 생체 조직 침투 증강용 조성물인 0.1v/v% Triton X-100 및 1M urea를 생체 조직 염색 시 함께 사용할 경우 염료(dye)의 침투를 증가시켜 발색이 더 선명하게 나타나는 것을 확인하였다. As a result, 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.
전술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야 한다.The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.
본 발명에 따른 Triton X-100 및 urea를 유효성분으로 포함하는 생체조직 침투 증강용 조성물은 생체 조직의 구조적 이미지를 보다 자세하게 관찰할 수 있고 관찰하기 어려운 조직의 변화를 분석할 수 있어 다양한 질환의 원인을 규명하고 치료법을 찾아내는 데 유용하게 이용 가능할 것으로 기대된다.The composition for enhancing tissue penetration of Triton X-100 and urea according to the present invention 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.

Claims (7)

  1. 트리톤 X-100(Triton X-100) 및 우레아(Urea)를 유효성분으로 포함하는 생체조직 침투 증강용 조성물.Triton X-100 (Triton X-100) and urea (Urea) comprising a composition for enhancing biological tissue penetration.
  2. 제1항에 있어서,The method of claim 1,
    상기 Triton X-100의 농도는 0.01 내지 0.5 v/v%인 것을 특징으로 하는, 생체조직 침투 증강용 조성물.The concentration of the Triton X-100 is characterized in that from 0.01 to 0.5 v / v%, biological tissue penetration enhancer composition.
  3. 제1항에 있어서,The method of claim 1,
    상기 Urea의 농도는 0.1 내지 5M인 것을 특징으로 하는, 생체조직 침투 증강용 조성물.The concentration of the Urea is characterized in that 0.1 to 5M, biological tissue penetration enhancer composition.
  4. 제1항에 있어서,The method of claim 1,
    상기 조성물은 항체 또는 염료(dye)의 침투를 증가시키는 것을 특징으로 하는, 생체조직 침투 증강용 조성물.The composition is characterized in that to increase the penetration of antibodies or dyes (dye), biological tissue penetration enhancer composition.
  5. 제4항에 있어서,The method of claim 4, wherein
    상기 염료(dye)는 블루 덱스트란(blue dextran)인 것을 특징으로 하는, 생체조직 침투 증강용 조성물.The dye (dye) is characterized in that the blue dextran (blue dextran), biological tissue penetration enhancer composition.
  6. 투명화된 생체조직에 제1항의 생체조직 침투 증강용 조성물을 항체 또는 염료(dye)와 함께 반응시키는 단계를 포함하는, 생체조직 침투 증강 방법.A method of enhancing biological tissue penetration, comprising reacting the composition for enhancing biological tissue penetration of claim 1 with an antibody or dye (dye).
  7. 트리톤 X-100(Triton X-100) 및 우레아(Urea)를 유효성분으로 포함하는 조성물의 생체조직 침투 증강 용도.The use of the composition containing the Triton X-100 and urea (Urea) as an active ingredient to enhance the penetration of biological tissues.
PCT/KR2019/003621 2018-03-29 2019-03-28 Composition for enhancing infiltration into biological tissues comprising triton x-100 and urea as active ingredients, and method of enhancing infiltration into biological tissues using same WO2019190216A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20010104441A (en) * 2000-04-28 2001-11-26 송우근 Method for expression and purification of hepatitis C virus core antigen and its hydrophilic fragment
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 (en) * 2000-04-28 2001-11-26 송우근 Method for expression and purification of hepatitis C virus core antigen and its hydrophilic fragment
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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