Use of mhc-ii expression in predicting efficacy of immunotherapy for gastric cancer

Abstract

The application discloses a use of MHC-II expression in prediction of curative effect of gastric cancer immunotherapy and belongs to the field of bioengineering. Specifically, the application comprises the following steps: detecting MHC-II expression in a gastric cancer biopsy sample; baking, dewaxing and gradient alcohol treating the gastric cancer biopsy sample slice; carrying out antigen repairing and blocking treatment; then adding diluted MHC Class II antibody for incubation, and then adding immunopotentiator and enhanced enzyme-labeled antibody for incubation; finally, completing immunostaining through DAB color development, staining liquid staining and counter blue treatment, mounting and observing. The MHC-II expression signal is clear and distinguishable, the staining effect is good, and through negative and positive expression interpretation and pathological remission evaluation, it is confirmed that the MHC-II expression is significantly related to pathological remission of a gastric cancer patient and plays an important role in prediction of curative effect of gastric cancer immunotherapy.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to the use of MHC-II expression in the prediction of the efficacy of immunotherapy for gastric cancer. BACKGROUND

[0002] Gastric and gastroesophageal junction (G / GEJ) cancer ranks fifth in newly diagnosed malignancies worldwide and fourth in cancer-related deaths. Despite the latest progress in multidisciplinary or multimodal therapy, the prognosis of patients with locally advanced disease remains poor, with a median overall survival (OS) of only 34.4 months and a 5-year OS rate of only 38.7%. Major histocompatibility complex (MHC) molecules, also known as human leukocyte antigen (HLA) molecules, have been found to be recognized by the immune system through major histocompatibility complexes (MHCs) to identify tumor cells. MHC is a cell surface receptor that binds to foreign peptides and presents them to T lymphocytes. Among them, MHC class II molecules are encoded by polymorphic MHC genes and consist of a non-covalent complex of alpha and beta chains. Helper T lymphocytes bind to antigen peptides presented by MHC class II molecules. MHC class II molecules bind to 13-18 amino acid antigen peptides. The accumulation of HLA-DM and -DO molecules in the endosomal / lysosomal compartment and on the surface of B cells regulates the binding of foreign peptides to class II molecules (HLA-DR) by maintaining a conformation that favors peptide exchange, which is essential for antigen presentation to CD4+ T lymphocytes, and the role of CD4+ T lymphocytes in antitumor immunity has also been increasingly emphasized. MHC-II is associated with a higher number of CD4+ and CD8+ tumor-infiltrating lymphocytes (TILs), lack of lymphatic vessel invasion, increased formation of tertiary lymphoid structures, upregulation of genes related to activation of the IFN-gamma pathway (including the CD274 gene encoding PD-L1), and higher levels of IFNG, IL2, and IL12 mRNA (Th1 cytokines). Therefore, MHC-II plays an important role in immunotherapy, and studies have shown that specific MHC-II expression in gastric cancer patients is closely related to immunotherapy.

[0003] MHC-II expression can be detected by immunohistochemical staining technology, which is a technology that uses the principle of antigen-antibody reaction to detect specific targets in various tissue samples in situ histology or cytology. With the development of antibody and antibody coupling technology, immunohistochemical staining is widely used, and the amount of target detected is large. As one of the practical and convenient in situ detection technologies, it has become a platform technology for basic research, pathological diagnosis and molecular target detection in clinical and basic medical fields. As an important target in the immune response of gastric malignancies, it is particularly important to determine the specific role of MHC-II expression in the prediction of the efficacy of immunotherapy for gastric cancer by immunohistochemical staining. SUMMARY

[0004] The purpose of the present application is to solve the problems existing in the prior art, and to provide the use of MHC-II expression in the prediction of the efficacy of gastric cancer immunotherapy by immunostaining.

[0005] To solve the above technical problems, the present application specifically provides the following technical solutions:

[0006] The present application discloses a gastric cancer immunotherapy efficacy prediction kit based on MHC-II expression, which comprises a staining solution; the staining solution comprises DAB developing solution, anti-blue solution and hematoxylin staining solution; the mass ratio of the DAB developing solution and the anti-blue solution is 1:0.4-0.8, and the mass ratio of the DAB developing solution and the hematoxylin staining solution is 1:0.8-1.5. The above-mentioned kit can accurately detect the expression of MHC-II in gastric cancer tissue by immunostaining technology, and provides a reliable basis for the prediction of the efficacy of immunotherapy.

[0007] Preferably, the hematoxylin staining solution comprises a dye and an auxiliary agent; the auxiliary agent comprises a mordant, and the mordant is citric acid, a polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid; the polycarboxylic acid is obtained by the reaction of maleic acid and itaconic acid. The use of citric acid, polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid can enhance the staining capacity and selective coloring capacity of the prepared hematoxylin staining solution, and can also make the staining background clearer, thereby obtaining better staining effect and staining intensity.

[0008] Preferably, the dye is hematoxylin.

[0009] Preferably, the auxiliary agent further comprises a metal mordant, a protective agent, an oxidizing agent and a solvent.

[0010] Preferably, the metal mordant is potassium aluminum sulfate, the protective agent is chloral hydrate, the oxidizing agent is sodium iodate, and the solvent is water.

[0011] Preferably, the anti-blue solution is obtained by mixing sodium bicarbonate in water, and the amount of sodium bicarbonate added to water is 0.0015-0.003 g / ml.

[0012] Preferably, the kit further comprises an antigen repair solution, which is obtained by mixing citric acid and sodium citrate in water, and the mass ratio of the use amount of citric acid to the use amount of sodium citrate is 1:6-8.5, and the amount of citric acid added to water is 0.0003-0.0005 g / ml.

[0013] Preferably, the kit further comprises a peroxidase inhibitor, and the peroxidase inhibitor is 1-3% hydrogen peroxide.

[0014] Preferably, the kit further comprises a blocking agent, and the blocking agent is sheep serum.

[0015] Preferably, the kit further comprises an antibody, which is MHC Class II recombinant rabbit monoclonal antibody and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

[0016] The application discloses a gastric cancer biopsy sample immunostaining method based on MHC-II expression.

[0017] S1: bake the tissue section at 70-80 DEG C for 15-25 min, soak in a deparaffinizing solution for 2-4 times, each time for 3-6 min, then soak in 100% alcohol, 95% alcohol, 80% alcohol and water for 2-5 min each time. Then, soak in an antigen repairing solution for high-pressure repairing for 60-120 s, and wash for 3-6 min. Dry the tissue section and draw a size. Then, add a peroxidase inhibitor and incubate for 8-15 min, and wash for 3-6 min. After adding a blocking agent and incubating for 8-15 min, a treated tissue section is obtained.

[0018] S2: add a diluted antibody to the treated tissue section, incubate at 0-5 DEG C for 5-10 h, and wash for 3-6 min. After washing, dry and soak in an immunoenhancer, place at 20-27 DEG C for 20 min, and wash for 3-6 min. Then, add a secondary antibody, incubate at 20-27 DEG C for 15-25 min, and wash for 3-6 min to obtain a tissue section compound.

[0019] S3: add a color developing agent to the tissue section compound, incubate at 20-27 DEG C for 2-5 min, and wash with water and dry. Add a counterstaining agent and soak for 1-5 min, wash with water and dry. Then, soak in a bluing agent for 1-5 min, and wash with water. Finally, soak in 100% alcohol, 95% alcohol, 80% alcohol and water for 2-5 min each time. Dry and seal with a sealing agent.

[0020] Preferably, the deparaffinizing solution is xylene, and the deparaffinizing solution is used in an amount sufficient to immerse the tissue section.

[0021] Preferably, the antigen repairing solution is obtained by mixing citric acid and sodium citrate in water, the mass ratio of the amount of citric acid to the amount of sodium citrate is 1:6-8.5, the amount of citric acid added in water is 0.0003-0.0005 g / ml, and the antigen repairing solution is used in an amount sufficient to immerse the tissue section.

[0022] Preferably, the peroxidase inhibitor is 1-3% hydrogen peroxide, and the peroxidase inhibitor is used in an amount sufficient to immerse the tissue section.

[0023] Preferably, the blocking agent is sheep serum, and the blocking agent is used in an amount sufficient to immerse the tissue section.

[0024] Preferably, the dilution antibody is diluted by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.01.

[0025] Preferably, the secondary antibody is an enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

[0026] Preferably, the dilution antibody and the secondary antibody are used in an amount sufficient to completely immerse the tissue section.

[0027] Preferably, the color developing agent is DAB color developing solution.

[0028] Preferably, the counterstain agent is hematoxylin staining solution.

[0029] Preferably, the counterstain agent is hematoxylin staining solution.

[0030] Preferably, the color developing agent, the counterstain agent, and the counterstain agent are used in an amount sufficient to completely immerse the tissue section.

[0031] Preferably, the washing reagent is PBS.

[0032] The present application discloses a preparation method of hematoxylin staining solution, comprising:

[0033] First, the dye and the metal mordant are added to the solvent, heated to boiling to obtain a dissolved solution, then the dyeing accelerator, the protective agent, and the oxidizing agent are dissolved to obtain the hematoxylin staining solution.

[0034] Preferably, the dyeing accelerator is citric acid, polycarboxylic acid, and 5-(hydroxymethyl)-2-furoic acid.

[0035] Preferably, the dye is hematoxylin.

[0036] Preferably, the metal mordant is potassium aluminum sulfate.

[0037] Preferably, the protective agent is chloral hydrate.

[0038] Preferably, the oxidizing agent is sodium iodate.

[0039] Preferably, the solvent is water.

[0040] Preferably, the mass ratio of the amount of the dye to the amount of the metal mordant is 1:43-53.

[0041] Preferably, the amount of the dye added to the solvent is 0.0008-0.0012g / ml.

[0042] Preferably, the mass ratio of the amount of the dye to the amount of the dyeing accelerator is 1:0.2-1.1.

[0043] Preferably, the mass ratio of the use amount of citric acid and the use amount of polycarboxylic acid in the mordant is 1:0.3-0.8, and the mass ratio of the use amount of citric acid and the use amount of 5-(hydroxymethyl)-2-furoic acid is 1:0.8-1.2.

[0044] Preferably, the mass ratio of the use amount of dye and the use amount of protective agent is 1:43-53.

[0045] Preferably, the mass ratio of the use amount of dye and the use amount of oxidizing agent is 1:0.17-0.22.

[0046] The application discloses a preparation method of polycarboxylic acid.

[0047] Maleic acid, itaconic acid and water are mixed to obtain a mixed solution, nitrogen is introduced, heating is performed to 55-70 DEG C, then potassium persulfate aqueous solution is dropped into the mixed solution, and reaction is performed for 0.8-1.5 h to obtain a transparent liquid, acetone is added into the transparent liquid to obtain a precipitate, the precipitate is extracted and dried to obtain polycarboxylic acid.

[0048] Preferably, maleic acid and itaconic acid are mixed in a mass ratio of 1:1-1.5.

[0049] Preferably, maleic acid and water are mixed in a mass ratio of 1:4-4.6.

[0050] Preferably, the potassium persulfate aqueous solution is obtained by combining potassium persulfate and water, and the mass ratio of the use amount of potassium persulfate and the use amount of water is 1:7.5-8.

[0051] Preferably, the mass ratio of the use amount of maleic acid and the use amount of potassium persulfate solution is 1:1.1-1.5.

[0052] More preferably, in the preparation of hematoxylin dyeing solution, alkyl polyglyceryl ether can be added on the basis of citric acid, polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid, and is used synergistically, so that the prepared hematoxylin dyeing solution can further improve the dyeing capacity of the hematoxylin dyeing solution, can present a clear contrast with the background, and better dyeing effect and dyeing strength can be obtained.

[0053] Preferably, the mass ratio of the use amount of hematoxylin and the use amount of alkyl polyglyceryl ether is 1:0.03-0.19.

[0054] The application discloses a preparation method of alkyl polyglyceryl ether.

[0055] The 3-chloro-1,2-propanediol is added into isopropyl alcohol and heated to 10-20℃, then the potassium hydroxide solution is added dropwise and reacted for 1-2h, after the reaction is completed, the glycidol is obtained by neutralization and filtration and drying. The glycidol and dodecanol are added into toluene, then the boron trifluoride ether is added, and the reaction is carried out at 30-40℃ for 1.5-2.5h under the condition of nitrogen, and the upper layer is separated to obtain the alkyl polyglyceryl ether.

[0056] Preferably, the amount of 3-chloro-1,2-propanediol added into isopropyl alcohol is 0.4-0.5g / ml.

[0057] Preferably, the potassium hydroxide solution is composed of potassium hydroxide and water, and the amount of potassium hydroxide in water is 0.35-0.45g / ml.

[0058] Preferably, the amount of 3-chloro-1,2-propanediol and the amount of potassium hydroxide solution are 1g:1-1.5ml.

[0059] Preferably, the mass ratio of the amount of glycidol used and the amount of dodecanol used is 1:0.8-1.

[0060] Preferably, the amount of glycidol added into toluene is 0.2-0.25g / ml.

[0061] Preferably, the mass ratio of the amount of glycidol used and the amount of boron trifluoride ether used is 1:0.0015-0.0025.

[0062] Compared with the prior art, the present application has the following beneficial effects:

[0063] The present application provides the use of MHC-II expression in the prediction of the efficacy of gastric cancer immunotherapy. First, the MHC-II expression in the gastric cancer biopsy sample is detected: the gastric cancer biopsy sample slice is baked, dewaxed, and treated with gradient alcohol; antigen repair and blocking treatment are performed; then the diluted MHC Class II antibody is added for incubation, followed by the addition of immune enhancer and enhanced enzyme-labeled antibody for incubation; finally, the immunostaining is completed by DAB color development, staining with staining solution, and counterstaining, and the slice is observed. The MHC-II expression signal is clear and distinguishable, and through pathological remission evaluation, it is confirmed that the MHC II expression is significantly related to the pathological remission of gastric cancer patients and plays an important role in the prediction of the efficacy of gastric cancer immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0064] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description are only exemplary, and for those skilled in the art, other drawings can be obtained from the provided drawings without creative labor.

[0065] Figure 1 Figure 2 is a graph of MHC-II expression in gastric cancer biopsy samples for efficacy prediction of immunotherapy.

[0066] Figure 2 Figure 3 is a graph of MHC-II expression in gastric cancer for efficacy prediction of immunotherapy. DETAILED DESCRIPTION

[0067] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative labor fall within the protection scope of the present application.

[0068] The concepts involved in the present application will be described below first with reference to the drawings. It should be noted that the following descriptions of the concepts are only for the purpose of making the content of the present application easier to understand, and do not represent a limitation on the protection scope of the present application; meanwhile, the embodiments in the present application and the features in the embodiments can be combined with each other without conflict. The present application will be described in detail below with reference to the drawings and in combination with the embodiments.

[0069] Embodiment 1

[0070] Gastric cancer biopsy sample immunostaining based on MHC-II expression

[0071] The tissue slices of patients with first diagnosis of gastric and gastroesophageal junction tumors were baked at 72℃ for 20 min, then immersed in xylene for 3 times, each time for 5 min, then placed in 100% alcohol, 95% alcohol, 80% alcohol and pure water for 3 min each. Then immersed in antigen repair solution for high pressure repair for 90 s, and washed with PBS for 5 min. After washing, the tissue slices were spun dry and the tissue size was drawn. Then 3% hydrogen peroxide was added and incubated for 10 min, and washed with PBS for 5 min. After washing, the treated tissue slices were obtained after blocking with sheep serum for 10 min. The antigen repair solution was obtained by mixing citric acid, sodium citrate and water, the mass ratio of the use amount of citric acid to the use amount of sodium citrate was 1:7.5, the amount of citric acid added to water was 0.0004 g / ml, and the use amount of 3% hydrogen peroxide and sheep serum was to completely immerse the tissue slices.

[0072] The diluted antibody was added to the treated tissue section, and incubated at 4°C for 8h, and washed with PBS for 5min. After washing, the tissue section was immersed in the immunoenhancing solution, and placed at 25°C for 20min, and washed with PBS for 5min. Then, the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody was added, and incubated at 25°C for 20min, and washed with PBS for 5min to obtain the tissue section complex. The diluted antibody was obtained by mixing phosphate buffer and MHC Class II recombinant rabbit monoclonal antibody at a volume ratio of 1:0.01, and the use amount of the diluted antibody and the enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody was complete immersion of the treated tissue section. The MHC Class II recombinant rabbit monoclonal antibody was purchased from Hangzhou Huaan Biotechnology Co., Ltd., and the product number was ET1704-13. The enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody and the immunoenhancing solution were obtained from a detection kit, and purchased from Beijing Zhongsu Jinqiao Biotechnology Co., Ltd., and the product number was PV-9001.

[0073] The DAB developing solution was added to the tissue section complex, and incubated at 25°C for 3min, and washed with water and spun dry. The hematoxylin dyeing solution was added to soak for 2min, and washed with water and spun dry. Then, the hematoxylin dyeing solution was immersed in the anti-blue solution to soak for 2min, and washed with water. Finally, the tissue section was immersed in 100% alcohol, 95% alcohol, 80% alcohol and pure water for 3min respectively. After drying, the tissue section was sealed with a sealing agent. The anti-blue solution was obtained by mixing sodium bicarbonate in water, and the amount of sodium bicarbonate added to water was 0.002g / ml. The use amount of the DAB developing solution, the hematoxylin dyeing solution and the anti-blue solution was complete immersion of the treated tissue section. The hematoxylin dyeing solution and the sealing agent were purchased from Ningbo Tongsheng Biological Technology Co., Ltd.

[0074] Example 2:

[0075] Preparation of the polycarboxylic acid: maleic acid, itaconic acid and water were mixed to obtain a mixed solution, and then nitrogen was introduced, and heated to 60°C. Then, the potassium persulfate aqueous solution was added dropwise into the mixed solution, and reacted for 1h to obtain a transparent liquid. The transparent liquid was added with acetone to obtain a precipitate, which was dried after filtration to obtain the polycarboxylic acid. The maleic acid and the itaconic acid were mixed at a mass ratio of 1:1.12, and the maleic acid and water were mixed at a mass ratio of 1:4.38. The potassium persulfate aqueous solution was obtained by combining potassium persulfate and water, and the mass ratio of the use amount of potassium persulfate to the use amount of water was 1:7.75. The mass ratio of the use amount of maleic acid to the use amount of potassium persulfate solution was 1:1.32.

[0076] Preparation of hematoxylin dye solution: first, hematoxylin and potassium aluminum sulfate were added to water, heated to boiling to obtain a dissolved solution, then citric acid, polycarboxylic acid, 5-(hydroxymethyl)-2-furoic acid, chloral hydrate and sodium iodate were added to the dissolved solution to obtain a hematoxylin dye solution. The mass ratio of the amount of hematoxylin used to the amount of potassium aluminum sulfate used was 1:50, the amount of hematoxylin added to water was 0.001 g / ml, the mass ratio of the amount of hematoxylin used to the amount of citric acid used was 1:0.3, the mass ratio of the amount of hematoxylin used to the amount of polycarboxylic acid used was 1:0.15, the mass ratio of the amount of hematoxylin used to the amount of 5-(hydroxymethyl)-2-furoic acid used was 1:0.3, the mass ratio of the amount of hematoxylin used to the amount of chloral hydrate used was 1:50, and the mass ratio of the amount of hematoxylin used to the amount of sodium iodate used was 1:0.2.

[0077] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: In this embodiment, the immunostaining of gastric cancer biopsy samples based on MHC-II expression is compared with Example 1, except that the hematoxylin dye solution is the hematoxylin dye solution prepared in this embodiment, and the other conditions and parameters are the same as in Example 1.

[0078] Example 3:

[0079] The preparation of polycarboxylic acid is the same as in Example 2.

[0080] Preparation of hematoxylin dye solution: In this embodiment, the preparation of hematoxylin dye solution is compared with Example 2, except that the mass ratio of the amount of hematoxylin used to the amount of polycarboxylic acid used is 1:0.1, and the other conditions and parameters are the same as in Example 2.

[0081] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: In this embodiment, the immunostaining of gastric cancer biopsy samples based on MHC-II expression is compared with Example 2, except that the hematoxylin dye solution used is the hematoxylin dye solution prepared in this embodiment, and the other conditions and parameters are the same as in Example 2.

[0082] Example 4:

[0083] The preparation of polycarboxylic acid is the same as in Example 2.

[0084] Preparation of hematoxylin dye solution: In this embodiment, the preparation of hematoxylin dye solution is compared with Example 2, except that the mass ratio of the amount of hematoxylin used to the amount of 5-(hydroxymethyl)-2-furoic acid used is 1:0.2, and the other conditions and parameters are the same as in Example 2.

[0085] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: In this embodiment, the immunostaining of gastric cancer biopsy samples based on MHC-II expression is compared with Example 2, except that the hematoxylin dye solution used is the hematoxylin dye solution prepared in this embodiment, and the other conditions and parameters are the same as in Example 2.

[0086] Example 5:

[0087] Preparation of polycarboxylic acid is the same as Example 2.

[0088] Preparation of alkyl polyglyceryl ether: 3-chloro-1,2-propanediol was added into isopropyl alcohol and heated to 15℃, then potassium hydroxide solution was added dropwise and reacted for 1.5h. After the reaction, glycidol was obtained by neutralization and filtration. Toluene was added into glycidol and dodecanol, then boron trifluoride ether was added under nitrogen condition, and reacted for 2h at 35℃. The upper layer was separated to obtain alkyl polyglyceryl ether. The amount of 3-chloro-1,2-propanediol added into isopropyl alcohol was 0.44g / ml, the potassium hydroxide solution was composed of potassium hydroxide and water, the amount of potassium hydroxide in water was 0.4g / ml, the amount ratio of 3-chloro-1,2-propanediol to potassium hydroxide solution was 1g:1.27ml, the mass ratio of the amount of glycidol to the amount of dodecanol was 1:0.9, the amount of glycidol added into toluene was 0.22g / ml, and the mass ratio of the amount of glycidol to the amount of boron trifluoride ether was 1:0.002.

[0089] Preparation of hematoxylin staining solution: hematoxylin and potassium aluminum sulfate were first added into water and heated to boiling to obtain a dissolved solution, then citric acid, polycarboxylic acid, 5-(hydroxymethyl)-2-furoic acid, alkyl polyglyceryl ether, chloral hydrate and sodium iodate were added into the dissolved solution to obtain hematoxylin staining solution. The mass ratio of the amount of hematoxylin to the amount of potassium aluminum sulfate was 1:50, the amount of hematoxylin added into water was 0.001g / ml, the mass ratio of the amount of hematoxylin to the amount of citric acid was 1:0.3, the mass ratio of the amount of hematoxylin to the amount of polycarboxylic acid was 1:0.15, the mass ratio of the amount of hematoxylin to the amount of 5-(hydroxymethyl)-2-furoic acid was 1:0.2, the mass ratio of the amount of hematoxylin to the amount of alkyl polyglyceryl ether was 1:0.08, the mass ratio of the amount of hematoxylin to the amount of chloral hydrate was 1:50, and the mass ratio of the amount of hematoxylin to the amount of sodium iodate was 1:0.2.

[0090] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: the difference between this example and Example 2 is that the hematoxylin staining solution used in this example is the hematoxylin staining solution prepared in this example, and other conditions and parameters are the same as those in Example 2.

[0091] Example 6:

[0092] Preparation of polycarboxylic acid is the same as Example 2.

[0093] Preparation of alkyl polyglyceryl ether is the same as Example 4.

[0094] Preparation of hematoxylin staining solution: the preparation of hematoxylin staining solution in this example is compared with that in Example 5, except that the mass ratio of the amount of hematoxylin used to the amount of alkyl polyglyceryl ether used is 1:0.05, and other conditions and parameters are the same as in Example 5.

[0095] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: this example is compared with Example 5, except that the hematoxylin staining solution used is the hematoxylin staining solution prepared in this example, and other conditions and parameters are the same as in Example 5.

[0096] Example 7:

[0097] The preparation of the polycarboxylic acid is the same as in Example 1.

[0098] The preparation of the alkyl polyglyceryl ether is the same as in Example 5.

[0099] Preparation of hematoxylin staining solution: the preparation of hematoxylin staining solution in this example is compared with that in Example 5, except that the mass ratio of the amount of hematoxylin used to the amount of alkyl polyglyceryl ether used is 1:0.12, and other conditions and parameters are the same as in Example 5.

[0100] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: this example is compared with Example 5, except that the hematoxylin staining solution used is the hematoxylin staining solution prepared in this example, and other conditions and parameters are the same as in Example 5.

[0101] Comparative Example 1:

[0102] Preparation of hematoxylin staining solution: the preparation of hematoxylin staining solution in this example is compared with that in Example 2, except that no polycarboxylic acid is used, and other conditions and parameters are the same as in Example 2.

[0103] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: this example is compared with Example 2, except that the hematoxylin staining solution used is the hematoxylin staining solution prepared in this example, and other conditions and parameters are the same as in Example 2.

[0104] Comparative Example 2:

[0105] Preparation of hematoxylin staining solution: the preparation of hematoxylin staining solution in this example is compared with that in Example 2, except that no 5-(hydroxymethyl)-2-furoic acid is used, and other conditions and parameters are the same as in Example 2.

[0106] Immunostaining of gastric cancer biopsy samples based on MHC-II expression: this example is compared with Example 2, except that the hematoxylin staining solution used is the hematoxylin staining solution prepared in this example, and other conditions and parameters are the same as in Example 2.

[0107] Experimental Example 1:

[0108] Gastric cancer patient and non-gastric cancer patient tissue sections were taken, and the immunostaining method for gastric cancer biopsy sample based on MHC-II expression in Example 1 was used for immunostaining. The tissue sections after immunostaining were observed, and according to the interpretation criteria and staining results, they were divided into negative and positive expression populations. The result interpretation criteria are as follows:

[0109] 0: no coloring or ≤10% of cancer cells present incomplete, weak cell membrane staining;

[0110] 1+: >10% of cancer cells present incomplete, weak cell membrane staining;

[0111] 2+: >10% of cancer cells present weak to moderate intensity complete cell membrane staining, and ≤10% of cancer cells present strong and complete cell membrane staining;

[0112] 3+: >10% of cancer cells present strong, complete and uniform cell membrane staining.

[0113] As shown in Figure 1 , immunohistochemical scores of 0 or 1+ are negative expression, and scores of 2+ or 3+ are positive expression, and the signal is clear and distinguishable.

[0114] Experimental Example 2:

[0115] Based on Experimental Example 1, pathological remission evaluation was performed on the negative and positive expression populations. 235 cases of neoadjuvant therapy (chemotherapy + immunotherapy) gastric cancer patients who underwent radical resection were analyzed using chi-square test. Pathological complete remission rate (pCR) refers to the absence of tumor cell residues (including lymph nodes, stage ypT0N0M0) under an optical microscope, and the main pathological remission rate (mPR) refers to tumor regression grade (TRG) being 0 or 1. The pathological remission evaluation criteria are shown in Table 1.

[0116] Table 1. Tumor pathological remission evaluation criteria

[0117]

[0118] The evaluation results are shown in Figure 2 , and the MHC II expression level is significantly related to pCR and mPR, showing the role of MHC II expression in gastric cancer pathological remission evaluation.

[0119] Experimental Example 3:

[0120] The color of the tissue section after staining with hematoxylin staining solution should generally show blue as the background. When the section shows blue-green color, the contrast is poor compared with the brownish positive signal shown after immunostaining of the tissue section of the gastric cancer patient, and the staining effect is not ideal. The tissue sections of non-gastric cancer patients were immunostained using the immunostaining method for gastric cancer biopsy samples based on MHC-II expression in Examples 1-7 and Comparative Examples 1-2. By observation, the proportion of blue color in the blue and blue-green colors in the tissue section was statistically calculated.

[0121] Table 2 Blue color proportion results

[0122]

[0123] As shown in Table 2, Example 1 compared with Example 2 shows that the hematoxylin staining solution prepared in Example 2 has better staining effect; Example 2 compared with Examples 3 and 4 shows that when the amount of polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid is reduced, the proportion of blue color in the tissue section after staining with the prepared hematoxylin staining solution is reduced, thereby reducing the staining effect; Example 5 compared with Example 2 shows that on the basis of using polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid, further adding alkyl polyglyceryl ether can play a synergistic promoting role, and the prepared hematoxylin staining solution can further increase the proportion of blue color in the tissue section, thereby improving the staining effect; Example 5 compared with Example 6 shows that when the amount of alkyl polyglyceryl ether is reduced to a certain extent, the prepared hematoxylin staining solution will reduce the proportion of blue color in the tissue section, i.e., reduce the staining effect; Example 2 compared with Comparative Examples 1 and 2 shows that polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid need to be used together, and the hematoxylin staining solution prepared by using polycarboxylic acid or 5-(hydroxymethyl)-2-furoic acid alone will affect the staining effect.

[0124] Experimental Example 4:

[0125] Staining intensity: The immunostained tissue sections in Examples 1-7 and Comparative Examples 1-2 were observed. "+" indicates positive staining result, and the more "+" indicates the stronger staining intensity.

[0126] Table 3 Immunostaining intensity results:

[0127]

[0128] The results are shown in Table 3. Compared with Example 1, Example 2 shows that the prepared hematoxylin dye has higher and better dyeing intensity. The dyeing intensity of Example 2 is stronger than that of Examples 3 and 4, indicating that the use of polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid in a reduced amount can reduce the effect of the prepared hematoxylin dye, thereby reducing the dyeing intensity. Compared with Example 2, Example 5 shows that on the basis of the use of polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid, further adding alkyl polyglyceryl ether can have a synergistic promotion effect, and the prepared hematoxylin dye can further improve the dyeing intensity. Compared with Example 5, Example 7 shows that the increase of the use amount of alkyl polyglyceryl ether to a certain extent can improve the dyeing intensity of the prepared hematoxylin dye. Compared with Comparative Examples 1 and 2, Example 2 shows that polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid need to be used together, and the use of polycarboxylic acid or 5-(hydroxymethyl)-2-furoic acid alone can inhibit the dyeing intensity of the prepared hematoxylin dye.

[0129] The above-described embodiments and / or implementations are merely used to illustrate the preferred embodiments and / or implementations of the present application, and are not intended to limit the embodiments of the present application in any form. Any person skilled in the art can make some changes or modifications to other equivalent embodiments without departing from the technical means disclosed in the content of the present application, and such changes or modifications should be considered as substantially the same technical or embodiments of the present application.

[0130] The principles and implementations of the present application are described by using specific examples. The above example is only used to help understand the method and its core idea of the present application. The above description is only the preferred embodiments of the present application. It should be pointed out that due to the limitation of language expression, there are infinite specific structures. For ordinary skilled persons in the technical field, some improvements, refinements or changes can be made without departing from the principles of the present application, and the above technical features can be combined in an appropriate manner. These improvements, refinements, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be considered as the protection scope of the present application.

Claims

1. A kit for predicting the efficacy of gastric cancer immunotherapy based on MHC-II expression, comprising a staining solution; the staining solution comprises DAB developing solution, counterstaining solution and hematoxylin staining solution; the mass ratio of the DAB developing solution to the counterstaining solution is 1:0.4-0.8, and the mass ratio of the DAB developing solution to the hematoxylin staining solution is 1:0.8-1.5; the hematoxylin staining solution comprises a dye and an auxiliary reagent; the auxiliary reagent comprises a mordant, and the mordant is citric acid, a polycarboxylic acid and 5-(hydroxymethyl)-2-furoic acid; the polycarboxylic acid is obtained by reacting maleic acid and itaconic acid; the kit further comprises an antibody, and the antibody is MHC Class II recombinant rabbit monoclonal antibody and enhanced enzyme-labeled goat anti-rabbit IgG polymer antibody.

2. The MHC-II expression-based gastric cancer immunotherapy efficacy prediction kit according to claim 1, characterized by, The dye is hematoxylin. 3.The MHC-II expression-based gastric cancer immunotherapy efficacy prediction kit of claim 1, wherein, The auxiliary reagent further comprises a metal mordant, a protective agent, an oxidizing agent and a solvent. 4.The MHC-II expression-based gastric cancer immunotherapy efficacy prediction kit of claim 3, characterized in that, The metal mordant is potassium aluminum sulfate, the protective agent is chloral hydrate, the oxidizing agent is sodium iodate, and the solvent is water.

5. The MHC-II expression-based gastric cancer immunotherapy efficacy prediction kit according to claim 1, characterized in that, The counterstaining solution is obtained by mixing sodium bicarbonate with water, and the amount of sodium bicarbonate added to water is 0.0015-0.003 g / ml. 6.The MHC-II expression-based gastric cancer immunotherapy efficacy prediction kit of claim 1, wherein, The kit further comprises an antigen retrieval solution, which is obtained by mixing citric acid and sodium citrate with water, the mass ratio of the amount of citric acid used to the amount of sodium citrate used is 1:6-8.5, and the amount of citric acid added to water is 0.0003-0.0005 g / ml. 7.The MHC-II expression-based gastric cancer immunotherapy efficacy prediction kit of claim 1, wherein, The kit further comprises a peroxidase inhibitor, and the peroxidase inhibitor is 1-3% hydrogen peroxide. 8.The MHC-II expression-based gastric cancer immunotherapy efficacy prediction kit of claim 1, wherein, The kit further comprises a blocking agent, and the blocking agent is goat serum.

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