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CN-121994558-A - Tissue decoloring, blood removing and decalcification composition and application thereof

CN121994558ACN 121994558 ACN121994558 ACN 121994558ACN-121994558-A

Abstract

The invention relates to a tissue decolorization and decalcification composition and application thereof, wherein the tissue decolorization and decalcification composition comprises a) a divalent cation chelating agent, b) an imidazole reagent, and c) water. The tissue decolorization, blood removal and decalcification composition can simultaneously realize decalcification, blood removal and decolorization of biological tissues, and can realize better decalcification and decolorization effects than the existing decalcification reagent and decolorization reagent respectively.

Inventors

  • YUAN KEXIN
  • GAO YIXIAO

Assignees

  • 北京清准医学科技有限公司
  • 清华大学

Dates

Publication Date
20260508
Application Date
20260109
Priority Date
20250810

Claims (14)

  1. 1. A tissue decolorizing and decalcifying composition comprising: a) A divalent cation chelator; b) An imidazole reagent which is an imidazole having an aminoalkyl group, and C) And (3) water.
  2. 2. The tissue decolorizing and decalcification composition according to claim 1, wherein said divalent cation chelator is selected from at least one of ethylenediamine tetraacetic acid, ethylenediamine succinic acid, iminodisuccinic acid, ethylenediamine di-o-phenylacetic acid, glutamic diacetic acid, methylglycine diacetic acid, nitrilotriacetic acid, and biologically acceptable salts thereof.
  3. 3. The tissue decolorizing and decalcification composition according to claim 1, wherein said aminoalkyl group is located on the nitrogen atom at position 1 or the carbon atom at position 2 of the imidazole ring, preferably said aminoalkyl group is a C1-6 aminoalkyl group.
  4. 4. The tissue decolorizing and decalcifying composition according to claim 3, wherein the imidazole reagent is at least one selected from the group consisting of 1- (3-aminopropyl) imidazole, 1- (2-aminoethyl) imidazole, 1- (aminomethyl) imidazole, 2- (3-aminopropyl) imidazole, 2- (2-aminoethyl) imidazole, and 2- (aminomethyl) imidazole.
  5. 5. The tissue decolorizing and decalcification composition according to claim 1, wherein said imidazole agent further comprises an alkyl group, preferably said alkyl group is a C1-6 alkyl group, preferably one of said aminoalkyl and alkyl groups is located on the nitrogen atom at position 1 of the imidazole ring and the other is located on the carbon atom at position 2.
  6. 6. The tissue decolorizing and decalcification composition according to claim 5, wherein said imidazole reagent is at least one selected from the group consisting of 1- (3-aminopropyl) -2-methyl-imidazole, 1- (2-aminoethyl) -2-methyl-imidazole, 1- (aminomethyl) -2-methyl-imidazole, 1-methyl-2- (aminomethyl) imidazole, 1-methyl-2- (2-aminoethyl) imidazole, 1-methyl-2- (3-aminopropyl) imidazole and 1-methyl-2- (2-aminopropyl) imidazole.
  7. 7. The tissue decolorization, dealkylation and decalcification composition according to any one of claims 1 to 6, wherein the pH of the tissue decolorization, dealkylation and decalcification composition is in the range of 7.5 to 11.0.
  8. 8. A tissue decolorization, dealkylation and decalcification composition according to any one of claims 1 to 6, wherein the content of divalent cation chelator in the tissue decolorization, dealkylation and decalcification composition is 5% to 20%, preferably 10% to 20% in w/v.
  9. 9. A tissue decolorization and decalcification composition according to any one of claims 1 to 6, wherein said imidazole-based agent is present in said tissue decolorization and decalcification composition in an amount of 5% to 40%, preferably 10% to 35% w/v.
  10. 10. The tissue decolorization and decalcification composition according to any one of claims 1 to 6, wherein said tissue decolorization and decalcification composition further comprises an additive selected from at least one of a pH adjustor, a permeation enhancer, and an antioxidant, and preferably, the content of said additive in said tissue decolorization and decalcification composition is 5% w/v or less.
  11. 11. The tissue decolorization and decalcification composition according to claim 10, wherein said tissue decolorization and decalcification composition comprises at least one pH adjustor selected from the group consisting of 4-hydroxyethylpiperazine ethanesulfonic acid ‌, triethanolamine, tris-HCl, preferably, said pH adjustor is at a concentration of 0.001M to 0.1M.
  12. 12. The tissue decolorization and decalcification composition according to claim 10, wherein said tissue decolorization and decalcification composition comprises at least one permeation enhancer selected from sodium deoxycholate and triton x-100, preferably said permeation enhancer is at a concentration of 0.1% w/v to 8% w/v.
  13. 13. The tissue decolorization and decalcification composition according to claim 10, wherein said tissue decolorization and decalcification composition comprises at least one antioxidant selected from potassium metabisulfite, mercaptoethanol and mercaptoethylamine, preferably said antioxidant is at a concentration of 0.02% w/v to 2% w/v.
  14. 14. A method for decolorizing and decalcifying biological tissue, comprising the steps of: 1) Obtaining a biological tissue sample from a donor, and 2) Treating the biological tissue sample with the tissue decolorizing and decalcifying composition of any one of claims 1 to 13, preferably the treatment time in step 2) is 30 minutes to 24 hours.

Description

Tissue decoloring, blood removing and decalcification composition and application thereof Technical Field The invention relates to the field of tissue treatment, in particular to a tissue decoloring, blood removing and decalcification composition and application thereof. Background The novel three-dimensional biology technology is combined with a thick tissue slice technology, a thick tissue fluorescent marking technology, a tissue transparentizing technology and a three-dimensional fluorescent imaging technology to obtain microscopic three-dimensional images of biological tissues. Compared with a section with a thickness of a few microns observed by two-dimensional histology, the three-dimensional histology can be used for observing a large number of tissue cells at a comprehensive and real visual angle, so that the judgment on the histopathological mechanism is more accurate and comprehensive, and the research on the drug mechanism and physiological phenomenon is more detailed and micro, so that the development and optimization of the three-dimensional histology imaging technology has great value in the fields of clinical diagnosis, scientific research, drug development and the like. In order to perform a treatment such as slicing and transparentizing on a tissue containing bones or pathological calcification foci, decalcification is generally required, the conventional decalcification agent is usually subjected to a decalcification treatment by a calcium ion chelating agent under a neutral condition, and the next operation can be performed after the bone tissue is softened, so that the decalcification efficiency and the decalcification effect are limited. On the other hand, blood-containing tissues such as human specimens or animal spleens, red bone marrow and the like cannot be perfused or red blood cells in the blood-containing tissues cannot be removed, and samples with severe blood staining can have good tissue transparentization effect or reduce the strong interference of strong autofluorescence of blood on fluorescence imaging after blood removal. The existing blood removing reagent has limited efficacy and cannot be used for effectively removing pigments in the tissues in an express way. In addition, tissues such as cardiac muscle and skeletal muscle contain myoglobin, and tissues such as liver and kidney contain more pigment components such as flavins and lipofuscins, which also affect the effects of tissue transparentization and fluorescence imaging. Therefore, it is necessary to develop a reagent capable of comprehensively realizing decalcification, decolorization and exsanguination of tissues, which has great application prospect in the aspects of biological imaging, in particular three-dimensional histological imaging. Disclosure of Invention In order to overcome the above problems of the prior art, a main object of the present invention is to provide a novel tissue decolorizing and decalcification composition, which can simultaneously realize decalcification, decalcification and decolorization of biological tissues, thereby greatly improving the treatment efficiency and realizing better decalcification and decolorization effects than the existing decalcification and decolorization reagents, respectively. The inventors of the present invention have unexpectedly found that decalcification, and decolorization of biological tissues can be simultaneously achieved by combining a divalent cation chelator and an imidazole-type reagent having an aminoalkyl group, and that even better effects than the existing decalcification reagent and decolorization reagent can be achieved, thereby completing the present invention. The tissue decoloring, bleeding and decalcification composition is used for decalcification, bleeding and decoloring of biological tissue materials, and is more beneficial to imaging of the processed biological tissue, in particular to three-dimensional histological imaging. Accordingly, in one aspect, the present invention provides a tissue decolorizing and decalcification composition comprising a) a divalent cation chelator, b) an imidazole agent having an aminoalkyl group, and c) water. In some embodiments, the divalent cation chelator is a calcium ion chelator comprising at least one selected from ethylenediamine tetraacetic acid, ethylenediamine succinic acid, iminodisuccinic acid, ethylenediamine di-o-phenylacetic acid, glutamic diacetic acid, methylglycine diacetic acid, nitrilotriacetic acid, and biologically acceptable salts thereof, preferably at least one selected from ethylenediamine tetraacetic acid, sodium ethylenediamine tetraacetic acid, ethylenediamine succinic acid, and sodium ethylenediamine succinic acid. In some embodiments, the aminoalkyl group may be located on the nitrogen atom at position 1 of the imidazole ring, or may be located on a carbon atom at position 2. In a preferred embodiment, the imidazole-based reagent is an imidazole having a C1-6 aminoalkyl group.