A TIPE3 immunohistochemical detection kit and its use method and application

Abstract

The present invention provides a TIPE3 immunohistochemical detection kit and a method and application thereof, belonging to the technical field of tumor detection. The present invention has found that TIPE3 expression is significantly increased in pancreatic cancer, is closely related to the invasion and metastasis potential of the tumor, and is an independent prognostic risk factor for pancreatic cancer patients. Therefore, the purpose of helping to judge the malignancy of pancreatic cancer and the prognosis of patients can be achieved by detecting the expression of TIPE3, thereby providing a TIPE3 immunohistochemical detection kit, which can effectively predict the malignancy and metastasis potential of pancreatic cancer, and perform recurrence and metastasis risk classification for pancreatic cancer patients; at the same time, the prognosis of patients can also be evaluated, thereby helping to improve the survival rate of pancreatic cancer patients, and providing an important scientific basis for the prevention and treatment of pancreatic cancer in clinical practice, so it has good clinical application value and social benefits.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor detection, and in particular relates to a TIPE3 immunohistochemical detection kit and a use method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Pancreatic cancer is one of the most malignant digestive system tumors. It develops insidiously, progresses rapidly, has a very poor prognosis, and the incidence rate is almost equal to the mortality rate. According to the latest statistics from the American Cancer Society (ACS), the expected incidence rate of pancreatic cancer ranks second among digestive system tumors, while the mortality rate ranks fourth among all malignant tumors. It is expected that by 2030, pancreatic cancer will become the second largest killer among all malignant tumors.

[0004] Although the diagnosis and treatment of pancreatic cancer are constantly being updated and improved, the prognosis of patients has not improved significantly in the past four decades, with a 5-year survival rate of only about 6%, making it the malignant tumor with the worst prognosis. Therefore, effective joint detection is urgently needed in the clinic to improve the overall treatment effect. In addition, the malignancy of pancreatic cancer can often only be judged based on the TNM staging of pancreatic cancer. The better the understanding of pancreatic cancer, the more conducive it is to treat patients.

[0005] The tumor necrosis factor alpha-induced protein 8 (TNFAIP8 or TIPE) family has four members: TIPE, TIPE1, TIPE2 and TIPE3, which are highly homologous in structure. TIPE3 was discovered in 2008 [see: Sun, H., et al. TIPE2, a negative regulator of innate and adaptive immunity that maintains immune homeostasis [J]. Cell. 2008, 133 (3): 415-426.]. Related studies have shown that TIPE3 is a transporter of phosphoinositide second messengers, which affects the growth, proliferation, invasion and migration of tumor cells by activating the phosphoinositide 3-kinase / protein kinase B (PI3K / AKT) pathway [Moniz, L., et al. A new TIPE of phosphoinositide regulator in cancer [J]. Cancer Cell. 2014, 26 (4): 443-444.].

[0006] Immunohistochemistry (IHC) detection uses the principle of specific binding of antigen and antibody. Through chemical reaction, the color developer (fluorescein, enzyme, metal ion, isotope) of the labeled antibody is used to determine the antigen (peptide and protein) in the tissue or cell, and to study its location, qualitative and relative quantitative. For example, the enzyme is connected to the antibody by covalent bond to make an enzyme-labeled antibody, and then the enzyme's specific catalytic effect on the substrate is used to generate colored insoluble products or particles with a certain electron density. Various antigen components are located on the cell surface or in the cell under an ordinary microscope or electron microscope. At present, immunohistochemical indirect staining is usually used for experiments. This technology can be used to detect various antigen substances at the tissue level or cell level, such as proteins, peptides, enzymes, hormones, pathogens and receptors. Because immunohistochemistry has the characteristics of strong specificity, high sensitivity, accurate positioning, and can organically combine morphological research with functional research, this new technology has been widely used in many fields of biological and medical research. In pathological research, the role and significance of immunohistochemistry are even more important. The inventors found that there is currently no report on the method of using immunohistochemistry to detect TIPE3 to evaluate the malignancy of pancreatic cancer and the prognosis of patients. Summary of the invention

[0007] Based on the above-mentioned deficiencies of the prior art, the present invention provides a TIPE3 immunohistochemical detection kit and its use method and application. The present invention has found that TIPE3 expression is significantly increased in pancreatic cancer, which is closely related to the potential for tumor invasion and metastasis, and is an independent prognostic risk factor for pancreatic cancer patients. Therefore, the purpose of helping to judge the malignancy of pancreatic cancer and the prognosis of patients can be achieved by detecting the expression of TIPE3, thereby providing a TIPE3 immunohistochemical detection kit, which has good practical application value.

[0008] The first aspect of the present invention provides a use of TIPE3 as a diagnostic marker in the preparation of pancreatic cancer-related detection products.

[0009] Among them, the pancreatic cancer-related detection products include the TIPE3 immunohistochemistry detection kit, which can be used to determine the malignancy of pancreatic cancer and the prognosis of patients.

[0010] The second aspect of the present invention provides a TIPE3 immunohistochemical detection kit, which comprises at least the following components: a primary antibody, a secondary antibody, a reagent for inactivating peroxidase and biotin in tissue sections, a colorimetric reagent that reacts with horseradish peroxidase and develops color, and a reagent that specifically stains cell nuclei in tissues;

[0011] The primary antibody is specifically a TIPE3-specific antibody prepared against human TIPE3 antigen, such as a rabbit anti-human TIPE3 polyclonal antibody, etc., which is not specifically limited here.

[0012] Correspondingly, the secondary antibody only needs to match the species, class or subclass of the primary antibody. For example, when the primary antibody is a rabbit anti-human TIPE3 polyclonal antibody, the secondary antibody can be goat anti-rabbit IgG (goat anti-rabbit IgG-HRP).

[0013] The third aspect of the present invention provides a method for using the above-mentioned TIPE3 immunohistochemical detection kit for tissue section immunohistochemical staining diagnosis, the method comprising:

[0014] S1. Take the tissue to be tested from the subject and fix it to make paraffin slices;

[0015] S2, the treated paraffin sections were reacted with primary antibody and secondary antibody successively;

[0016] S3. After the reaction is completed, the sections are washed, stained with DAB, counterstained with hematoxylin, dehydrated, transparently sealed, and observed under a microscope.

[0017] A fourth aspect of the present invention provides the use of the above-mentioned TIPE3 immunohistochemical detection kit and / or method for use in tumor detection.

[0018] Wherein, the tumor is pancreatic cancer;

[0019] The tumor detection specifically includes: judging the malignancy of pancreatic cancer, metastasis potential and patient prognosis (including the survival rate of pancreatic cancer patients).

[0020] Beneficial technical effects of one or more of the above technical solutions:

[0021] The above technical scheme is based on the fact that the higher the intensity and the wider the range of TIPE3 immunohistochemical staining of pancreatic cancer tissues in pancreatic cancer patients, the higher the malignancy of pancreatic cancer; conversely, the lower the malignancy, thereby preparing a TIPE3 immunohistochemical detection kit, which can effectively distinguish pancreatic cancer tissues from normal tissues, and can also provide new diagnostic and classification basis for personalized treatment of pancreatic cancer.

[0022] The above-mentioned kit can effectively predict the malignancy and metastatic potential of pancreatic cancer, and grade the risk of recurrence and metastasis of pancreatic cancer patients; it can also evaluate the patient's prognosis, thereby helping to improve the survival rate of pancreatic cancer patients and provide an important scientific basis for the clinical prevention and treatment of pancreatic cancer. Therefore, it has good clinical application value and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0024] Figure 1 is the survival curve of patients with high and low TIPE3 expression in the embodiment of the present invention;

[0025] Figure 2 This is an IHC staining image of normal tissue surrounding pancreatic cancer in an embodiment of the present invention;

[0026] Figure 3 is an IHC staining diagram of pancreatic cancer tissue in an embodiment of the present invention;

[0027] Figure 4 is an IHC staining image of pancreatic cancer tissue with negative lymph node metastasis in an embodiment of the present invention;

[0028] Figure 5 is an IHC staining image of pancreatic cancer tissue with positive lymph node metastasis in an embodiment of the present invention;

[0029] Figure 6 is an IHC staining image of lymph node tissue without metastasis in the embodiment of the present invention;

[0030] Figure 7 is an IHC staining image of lymph node tissue with metastasis in an embodiment of the present invention;

[0031] Figure 8 : This is the ROC curve of using the method to predict whether pancreatic cancer patients have lymph node metastasis in the embodiment of the present invention. DETAILED DESCRIPTION

[0032] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0033] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0034] As mentioned above, there is no report on the method of using immunohistochemical detection of TIPE3 to evaluate the malignancy of pancreatic cancer and the prognosis of patients. The inventors found that the expression of TIPE3 protein in pancreatic cancer tissue by immunohistochemical staining was significantly higher than that in the surrounding non-cancerous tissue by immunohistochemical staining of pancreatic cancer tissue by TIPE3 immunohistochemical staining of pancreatic cancer patients with lymph node metastasis was higher than that of pancreatic cancer patients without lymph node metastasis. In addition, the survival rate of pancreatic cancer patients with high expression of TIPE3 is lower than that of pancreatic cancer patients with low expression of TIPE3. Therefore, the higher the intensity and the wider the range of TIPE3 immunohistochemical staining of pancreatic cancer tissue in pancreatic cancer patients, the higher the malignancy of pancreatic cancer; otherwise, the lower the malignancy. Based on the above research results, the present invention is proposed.

[0035] In a specific embodiment of the present invention, there is provided a use of TIPE3 as a diagnostic marker in the preparation of pancreatic cancer-related detection products.

[0036] Among them, the pancreatic cancer-related detection products include the TIPE3 immunohistochemistry detection kit, which can be used to determine the malignancy of pancreatic cancer and the prognosis of patients.

[0037] In another specific embodiment of the present invention, a TIPE3 immunohistochemical detection kit is provided, and the TIPE3 immunohistochemical detection kit comprises at least the following components: a primary antibody, a secondary antibody, a reagent for inactivating peroxidase and biotin in tissue sections, a color developing reagent that reacts with horseradish peroxidase and develops color, and a reagent that specifically stains cell nuclei in tissues;

[0038] The primary antibody is specifically a TIPE3-specific antibody prepared against human TIPE3 antigen, such as a rabbit anti-human TIPE3 polyclonal antibody, etc., which is not specifically limited here.

[0039] Correspondingly, the secondary antibody only needs to match the species, class or subclass of the primary antibody. For example, when the primary antibody is a rabbit anti-human TIPE3 polyclonal antibody, the secondary antibody can be goat anti-rabbit IgG (goat anti-rabbit IgG-HRP).

[0040] The reagent for inactivating peroxidase and biotin in the tissue slice can be a 0.3% H2O2 solution.

[0041] The color developing reagent that reacts with horseradish peroxidase and develops color can be a DBA solution.

[0042] The reagent that specifically stains cell nuclei in tissues may be hematoxylin.

[0043] In another specific embodiment of the present invention, the TIPE3 immunohistochemical detection kit may further include a fixative, a dewaxing hydrating agent, a serum blocking agent, a buffer, an antibody diluent, a dehydrating transparent agent, and the like.

[0044] Wherein, the fixative may be neutral buffered 4% paraformaldehyde solution, Bouin's solution or mDF solution.

[0045] The serum blocking agent is generally an antibody in the serum of an animal from the same source as the secondary antibody, which can bind to the sites in the tissue that can bind to the secondary antibody in advance; calf serum, BSA, sheep serum, etc. can also be used, but they cannot be from the same source as the primary antibody.

[0046] The buffer may be PBS buffer.

[0047] PBS can be used as the antibody diluent, or a special antibody diluent to which sodium azide preservative, BSA stabilizer and other components are added to facilitate multiple recycling of antibodies can be used.

[0048] The dewaxing hydrating agent and the dehydrating transparent agent are both xylene and ethanol (including anhydrous ethanol, 95% ethanol, 85% ethanol and 75% ethanol), but the order of use is completely opposite.

[0049] In another specific embodiment of the present invention, a method for using the above-mentioned TIPE3 immunohistochemical detection kit for tissue section immunohistochemical staining diagnosis is provided, and the method comprises:

[0050] S1. Take the tissue to be tested from the subject and fix it to make paraffin slices;

[0051] S2, the treated paraffin sections were reacted with primary antibody and secondary antibody successively;

[0052] S3. After the reaction is completed, the sections are washed, stained with DAB, counterstained with hematoxylin, dehydrated, transparently sealed, and observed under a microscope.

[0053] Wherein, in step S1, the tissue to be tested of the subject may be pancreatic tissue (such as pancreatic cancer tissue).

[0054] In step S2, the paraffin slice treatment process includes: dewaxing and hydrating the prepared paraffin slice, performing antigen repair, then inactivating peroxidase and biotin and performing serum blocking.

[0055] In step S3, the microscopic observation includes: randomly selecting three fields of view for each sample, with about 500 cells in each group; performing semi-quantitative scoring according to the intensity of hematoxylin staining (0, negative; 1, +; 2, ++; 3, +++), and dividing into four levels according to the percentage of positive cells (<1%, 0 points; 1-25%, 1 point; 26-50%, 2 points; 60-75%, 3 points; 76-100%, 4 points); combining the two scores of each sample to obtain the final TIPE2 expression score (IHC sum score). The cutoff points for defining the expression level are: 0-3 points, low expression; 4-8 points, high expression.

[0056] In another specific embodiment of the present invention, there is provided application of the above-mentioned TIPE3 immunohistochemical detection kit and / or method of use in tumor detection.

[0057] Wherein, the tumor is pancreatic cancer;

[0058] The tumor detection specifically includes: judging the malignancy of pancreatic cancer, metastasis potential and patient prognosis (including the survival rate of pancreatic cancer patients).

[0059] The present invention is further explained by the following examples, but they are not intended to limit the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0060] Example

[0061] The method of using the TIPE3 immunohistochemistry test kit to evaluate the malignancy of pancreatic cancer and the prognosis of patients includes five parts: sample processing, initial test, antibody incubation, color development, observation and conclusion drawing, which are specifically divided into the following steps:

[0062] (1) Tissue fixation: After taking pancreatic cancer samples from patients, fixative treatment is required to maintain the inherent morphology and structure of cells, preserve the antigenicity of tissue cells, remove lipids that hinder antigen-antibody binding, and make it easier for antigen-antibody conjugates to obtain good staining results. Neutral buffered 4% paraformaldehyde solution is commonly used (Bouin's solution, mDF solution, etc. can also be used). The tissue fixation time is best within 12 hours and generally should not exceed 24 hours. Because as the fixation time increases, the detection intensity of tissue antigens will gradually decrease.

[0063] (2) Preparation: Paraffin slices can be made. Paraffin slices are simple to store, have beautiful structures, and can be stored for a long time. First, use gradient ethanol (from low to high, usually starting from 50% alcohol, through 70%, 85%, 95% to pure alcohol) for dehydration, then use a transparent agent to impregnate the tissue, then completely immerse the tissue in wax, and finally slice it. Generally, the paraffin slice is sliced ​​about 5μm. After the slices are completed, they are pasted (fished out) and baked. A thin layer of protein glycerol can be applied on the slide, and then the wax slice can be flattened in warm water (about 45°C), fished out and laid on the slide, excess water can be absorbed with filter paper, and the slide can be placed in a 45°C incubator to dry.

[0064] (3) Dewaxing and hydration of paraffin sections: In order for antibodies and other reagents to fully react with antigens in tissues, pancreatic cancer sample sections need to be washed in xylene and gradient ethanol (from high to low). Xylene I for 10 minutes, xylene II for 10 minutes; anhydrous ethanol for 5 minutes, 95% ethanol for 5 minutes, 85% ethanol for 5 minutes, 75% ethanol for 5 minutes; phosphate buffered saline (PBS) for 2 times, and placed on a shaker.

[0065] (4) Antigen repair: During the fixation process of formaldehyde or paraformaldehyde, some antigens in the tissue will undergo cross-linking between proteins and the blocking effect of aldehyde groups, thus losing their antigenicity; antigen repair can re-expose the antigenic determinant clusters in the cell, thereby improving the antigen detection rate. Commonly used repair methods can be divided into three types from strong to weak: high-pressure repair, microwave repair, and pancreatic enzyme repair. Generally, the tissue section can be placed in the repair solution, and then microwave repaired on medium heat for 6 minutes*4 times. Note that it should be cooled naturally for about 30 minutes after microwave repair.

[0066] (5) Inactivation of peroxidase and biotin: If the color development system is a peroxidase (HRP) system, or in the traditional ABC method and SP method, the immunohistochemical reaction results are easily interfered by endogenous peroxidase and biotin. In order to prevent non-specific color development, hydrogen peroxide and avidin must be used for inactivation. In addition, some tissues have high endogenous peroxidase content and need to be inactivated. The sample slide can be placed in 0.3% H2O2 and incubated for 10 minutes. Then wash twice in PBS solution, 10 minutes each time.

[0067] (6) Serum blocking: Serum blocking is used to exclude nonspecific binding sites and nonspecific staining. The blocking serum is generally the antibody in the serum of the animal from the same source as the secondary antibody, which can bind to the sites in the tissue that can bind to the secondary antibody in advance; calf serum, BSA, sheep serum, etc. can also be used, but they cannot be the same source as the primary antibody. It can be kept at room temperature for 10-30 minutes to prevent excessive blocking.

[0068] (7) Primary and secondary antibody treatment:

[0069] There are several primary antibody incubation temperatures: 4°C, room temperature, and 37°C, with 4°C incubation having the best effect; incubation time: This is related to temperature and antibody concentration. It can be incubated at 37°C for 1-2 hours, while at 4°C it needs to be incubated overnight, and then taken out of the refrigerator and reheated at 37°C for 45 minutes.

[0070] The secondary antibody can be incubated at room temperature or 37°C for 30 min-1 h. The secondary antibody must match the species, class or subclass of the primary antibody.

[0071] In this embodiment, the primary antibody used for immunohistochemistry was synthesized by Wuhan Boster Biotechnology Co., Ltd. The designed and synthesized TIPE3 antigen epitope peptide contained 20 amino acids at the C-terminus (RPNLKRICEGINKLLDEKVL), the dilution concentration was 1:300, and the incubation time was overnight at 4°C; the secondary antibody was horseradish peroxidase-labeled goat anti-rabbit IgG (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).

[0072] PBS can be used as the antibody diluent, or a special antibody diluent to which sodium azide preservative, BSA stabilizer and other components are added to facilitate multiple recycling of antibodies can be used.

[0073] (8) Section cleaning (immersion, washing and rinsing): In order to prevent nonspecific staining caused by residual primary antibody, secondary antibody and other reagents, it is particularly important to appropriately strengthen the cleaning (extend the time and increase the number of times). The cleaning before primary antibody incubation can be: 3 minutes * 3 times, and the cleaning after primary antibody incubation can be: 5 minutes * 5 times.

[0074] (9) DAB color development: Add DBA solution, color development, observe the brown background under an optical microscope, and rinse with tap water to stop. The DAB color development time is not fixed. It is mainly controlled by the microscope. When the specific staining is strong and the background coloring is light, rinse.

[0075] (10) Counterstaining: Hematoxylin counterstaining (nuclear dye) is commonly used. The purpose is to form the cell outline, so as to better locate the target protein. After counterstaining, the slide is washed with running water, then placed in hydrochloric acid alcohol for a few seconds, washed with running water again, and finally placed in ammonia water to turn blue.

[0076] (11) Dehydration and transparent mounting: The dehydration and transparent process is the opposite of the dewaxing process. Wash the slides in gradient ethanol (from low to high) and xylene in turn: 75% ethanol for 5 minutes → 85% ethanol for 5 minutes → 95% ethanol for 10 minutes → anhydrous ethanol for 10 minutes → xylene for 10 minutes, remove the liquid, and dry at room temperature. For transparency, xylene II for 10 minutes and xylene I for 10 minutes can be used. For long-term storage, neutral gum is generally used for mounting. You can directly drop a drop of mounting liquid on the slide tissue, and then cover it with a coverslip, paying attention to the generation of bubbles. Let it dry for half a day.

[0077] (12) Observation under a microscope.

[0078] Immunohistochemical staining scoring method: Two experienced pathologists independently performed blind evaluation. When there was a large difference (score difference > 2), a third pathologist was required to analyze the results. Three fields of view were randomly selected for each sample under an optical microscope, with approximately 500 cells in each group. Semi-quantitative scoring was performed according to the intensity of hematoxylin staining (0, negative; 1, +; 2, ++; 3, +++), and the percentage of positive cells was divided into four levels (<1%, 0 points; 1-25%, 1 point; 26-50%, 2 points; 60-75%, 3 points; 76-100%, 4 points). The two scores of each sample were combined to obtain the final TIPE2 expression score (IHCsum score). The cutoff points for defining the expression level were: 0-3 points, low expression; 4-8 points, high expression.

[0079] Perform TIPE3 immunohistochemical detection on cancer tissues of pancreatic cancer patients, such as Figure 3 As shown in Figure 2, the expression of TIPE3 protein in pancreatic cancer tissues was significantly higher than that in surrounding non-cancerous tissues by immunohistochemical staining ( Figure 2 ). The immunohistochemical detection of TIPE3 in cancer tissues of pancreatic cancer patients with lymph node metastasis ( Figure 5 )TIPE3 protein expression was higher in pancreatic cancer patients without lymph node metastasis ( Figure 4 ). In addition, if Figure 1 As shown in the figure, our clinical data show that the survival rate of pancreatic cancer patients with high TIPE3 expression is lower than that of pancreatic cancer patients with low TIPE3 expression. In addition, lymph node metastasis is more likely to occur when the immunohistochemical staining score is 4 or above. In summary, the higher the intensity and the wider the range of TIPE3 immunohistochemical staining in the cancer tissue of pancreatic cancer patients, the higher the malignancy of pancreatic cancer; conversely, the lower the malignancy.

[0080] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to perform equivalent replacements on parts thereof. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention. Although the above describes the specific implementation methods of the present invention, it is not intended to limit the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solution of the present invention, various modifications or deformations that can be made by those skilled in the art without creative labor are still within the protection scope of the present invention.

Claims

1. Application of TIPE3 as a prognostic marker in the preparation of pancreatic cancer-related detection products; The pancreatic cancer-related detection products include a TIPE3 immunohistochemistry detection kit; the TIPE3 immunohistochemistry detection kit is used to determine the malignancy of pancreatic cancer and the prognosis of patients; the prognosis of patients is the survival rate of pancreatic cancer patients; The method for using the TIPE3 immunohistochemistry detection kit comprises: S1. Take the tissue to be tested from the subject and fix it to make paraffin slices; S2, the treated paraffin sections were reacted with primary antibody and secondary antibody successively; S3. After the reaction is completed, the slices are washed, stained with DAB, counterstained with hematoxylin, dehydrated, transparently sealed, and observed under a microscope; Wherein, in step S1, the tissue to be tested of the subject is pancreatic cancer tissue; In step S2, the paraffin slice treatment process includes: dewaxing and hydrating the prepared paraffin slice, performing antigen repair, then inactivating peroxidase and biotin and performing serum blocking; In step S3, the microscopic observation includes: randomly selecting three fields of view for each sample, with about 500 cells in each group; performing semi-quantitative scoring according to the intensity of hematoxylin staining: 0, negative; 1, +; 2, ++; 3, +++, and dividing into four levels according to the percentage of positive cells: <1%, 0 point; 1-25%, 1 point; 26-50%, 2 points; 60-75%, 3 points; 76-100%, 4 points; combining the two scores of each sample to obtain the final TIPE2 expression score; defining the cutoff point of the expression level as: 0-3 points, low expression; 4-8 points, high expression.

Images