Application of EphB3 gene as biomarker in preparation of Crohn disease detection product

Abstract

The invention discloses application of an EphB3 gene as a biomarker in preparation of a Crohn disease detection product. According to the present invention, the expression of the EphB3 gene in the Crohn disease patient is significantly up-regulated, such that the EphB3 gene can be adopted as the Crohn disease-related biomarker, can specifically detect the Crohn disease infection, and has extremely high accuracy. Meanwhile, according to the application of a reagent for detecting EphB3 receptor expression in preparing a reagent for identifying and diagnosing the Crohn disease, the expression quantity of EphB3 in the intestinal mucosa of a patient suffering from the Crohn disease is remarkably increased compared with the expression quantity of EphB3 in the intestinal mucosa of a patient suffering from enterophthisis and a patient suffering from primary intestinal lymphoma, and the difference has statistical significance (Plt; therefore, the detection of the expression of the EphB3 can be used for differential diagnosis of Crohn's disease, intestinal tuberculosis and primary intestinal lymphoma, and has extremely high specificity and accuracy.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to the application of EphB3 gene as a biomarker in the preparation of Crohn's disease detection products. Background Art

[0002] Crohn's disease (CD), also known as regional ileitis and granulomatous enteritis, is an intestinal inflammatory disease of unknown cause; this disease and chronic nonspecific ulcerative colitis are collectively referred to as inflammatory bowel disease (IBD). Crohn's disease can occur in any part of the gastrointestinal tract, but it is more common in the terminal ileum and right colon. The main symptoms are abdominal pain, diarrhea, and intestinal obstruction, and there are extraintestinal manifestations such as fever and nutritional disorders. The course of the disease is often prolonged, often recurring, and difficult to cure.

[0003] Crohn's disease has many similarities with intestinal tuberculosis (ITB) and primary intestinal lymphoma (PIL) in terms of clinical manifestations, laboratory tests, endoscopic examinations, imaging and even histology. However, the treatment plans for these three diseases are completely different. Misdiagnosis or delayed diagnosis may lead to the progression and deterioration of the disease, bringing greater psychological and economic burdens to patients and social medical care. Nowadays, it is still a major problem to quickly and accurately differentiate Crohn's disease from intestinal tuberculosis and primary intestinal lymphoma in clinical practice.

[0004] At present, the main means of distinguishing Crohn's disease from primary intestinal lymphoma is to construct a clinical diagnostic model. However, the formula of the model is generally complex and difficult to apply clinically. In the past, there were many means of distinguishing between Crohn's disease and intestinal tuberculosis, such as CT enterography and intestinal ultrasound. However, this can only obtain an indirect imaging manifestation, which requires observation of tissue granulomas. However, it is difficult to obtain qualified tissue specimens under clinical endoscopy, and the requirements for endoscopists are high. New diagnostic indicators such as ASCA and ANCA, but their research results are still controversial and need further research and clarification. It is urgent to develop rapid and accurate means of distinguishing Crohn's disease from intestinal tuberculosis and primary intestinal lymphoma.

[0005] EphB3 (ephrin B receptor 3) is a receptor for members of the ephrin B family (including ephrin B1, ephrin B2 and ephrin B3). It is known that there are significant differences in the expression of EphB3 in normal tissues and certain tumors and cancer cell lines, and it can be used as a marker for related cancers; however, there have been no reports on the application of EphB3 in the detection and diagnosis of Crohn's disease, especially in the differential diagnosis between Crohn's disease and intestinal tuberculosis and primary intestinal lymphoma. Summary of the invention

[0006] Based on this, the purpose of the present invention is to provide a Crohn's disease-related biomarker that can specifically detect Crohn's disease infection with high specificity and accuracy.

[0007] The first aspect of the present invention provides the use of EphB3 gene as a biomarker in the preparation of Crohn's disease detection products.

[0008] The second aspect of the present invention provides the use of a reagent for detecting the expression level of the EphB3 gene in the preparation of a Crohn's disease detection product.

[0009] In some of the embodiments, the detection product includes reagents using polymerase chain reaction technology, in situ hybridization technology, enzymatic mutation detection technology, chemical shearing mismatch technology, mass spectrometry analysis technology, gene chip technology or gene sequencing technology or a combination thereof.

[0010] In some of the embodiments, in the polymerase chain reaction technology, the polymerase chain reaction is any one of RT-PCR, immune PCR, nested PCR, fluorescent PCR, in situ PCR, membrane-bound PCR, anchored PCR, fixed PCR, in situ PCR, asymmetric PCR, long-distance PCR, parachute PCR, and gradient PCR.

[0011] The third aspect of the present invention provides the use of a reagent for detecting the amount of EphB3 receptors in the preparation of a Crohn's disease detection product.

[0012] In some embodiments, the detection product includes reagents using protein chip technology, ELSA or immunohistochemistry technology or a combination thereof.

[0013] In some embodiments, the test sample of the product is an intestinal tissue sample.

[0014] In some of the embodiments, a reagent for detecting the expression level of the EphB3 gene and / or the amount of the EphB3 receptor is used in the preparation of a differential diagnostic reagent for Crohn's disease, intestinal tuberculosis, or primary intestinal lymphoma.

[0015] A fourth aspect of the present invention provides a kit for detecting Crohn's disease, wherein the kit uses polymerase chain reaction technology to detect the EphB3 gene; or,

[0016] The kit uses immunohistochemical analysis technology to detect EphB3 receptor.

[0017] In some embodiments, the kit is based on the immunohistochemical analysis technology to collect EphB3 receptor data processing method, comprising the following steps:

[0018] S1, obtain intestinal tissue embedded sections, dewax, antigen retrieval, block endogenous peroxidase, block serum, and obtain pre-treated tissue sections;

[0019] S2, adding anti-EphB3 primary antibody to the pre-treated tissue section for immunobinding, incubating for 10-14 hours at a temperature of 2-6° C., then adding an enzyme-labeled secondary antibody for binding to the anti-EphB3 primary antibody for immunobinding, incubating for 40-60 minutes at a temperature of 22-28° C., to obtain an immunolabeled tissue section; the marker of the enzyme-labeled secondary antibody includes horseradish peroxidase and alkaline phosphatase;

[0020] S3, adding a color developing solution to the immunolabeled tissue section, counter-staining the cell nucleus, dehydrating and sealing the section, scanning to obtain a tissue image, analyzing the positive expression area ratio of the tissue image, and obtaining the expression amount data of EphB3.

[0021] In some embodiments, the antigen repair operation includes: placing the dewaxed intestinal tissue embedded section in a repair box containing a citric acid antigen repair buffer with a pH of 5.8-6.5, heating for 6-10 minutes to boiling, then heating at 40-70°C for 5-10 minutes, cooling naturally, and placing in a PBS buffer;

[0022] The operation of blocking endogenous peroxidase comprises: soaking in a hydrogen peroxide solution with a volume fraction of 1-5% as a blocking agent for 20-30 minutes;

[0023] The operation of blocking serum includes: using a BSA solution with a mass fraction of 1-5% as a blocking agent for blocking treatment for 25-35 minutes.

[0024] The inventors of the present invention have found in their research that the expression of the EphB3 gene is significantly upregulated in Crohn's disease patients, so the EphB3 gene can be used as a biomarker related to Crohn's disease, and can specifically detect Crohn's disease infection with extremely high accuracy. At the same time, in view of the difficulty of differential diagnosis of Crohn's disease, intestinal tuberculosis and primary intestinal lymphoma, which have the same clinical manifestations, it has been found that the expression of the EphB3 gene in the intestinal mucosa of Crohn's disease patients is significantly upregulated compared with the expression in the intestinal mucosa of intestinal tuberculosis patients and primary intestinal lymphoma patients, so the EphB3 gene can be used for the differential diagnosis of the above-mentioned diseases, and has important clinical diagnostic value and clinical significance for the early and rapid differential diagnosis of Crohn's disease infection.

[0025] In addition, the study found that detecting the amount of EphB3 receptors can also be used to detect Crohn's disease infection. The expression level of EphB3 receptors in the intestinal mucosa of Crohn's disease patients was significantly higher than that in the intestinal mucosa of patients with intestinal tuberculosis and primary intestinal lymphoma, and the differences were statistically significant (P<0.0001). Therefore, detecting the amount of EphB3 receptors can also be used for the differential diagnosis of Crohn's disease from intestinal tuberculosis and primary intestinal lymphoma, with extremely high specificity and accuracy, providing direction for the further development of rapid diagnostic kits for use in Crohn's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a comparative diagram of the expression of the EphB3 gene in Crohn's disease, intestinal tuberculosis and primary intestinal lymphoma in Example 1 of the present invention.

[0027] Figure 2 This is an immunohistochemical staining diagram of intestinal mucosal samples of patients with Crohn's disease, intestinal tuberculosis and primary intestinal lymphoma in Example 2 of the present invention.

[0028] Figure 3 The present invention Figure 2 Statistical graph of the percentage of EphB3-positive area.

[0029] Figure 4 3 is a ROC curve diagram for distinguishing Crohn's disease and intestinal tuberculosis in Example 2 of the present invention.

[0030] Figure 5 3 is a ROC curve diagram for distinguishing Crohn's disease and primary intestinal lymphoma in Example 2 of the present invention. DETAILED DESCRIPTION

[0031] The technical scheme of the present invention is further illustrated by specific examples below. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0032] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0033] Example 1

[0034] Expression of EphB3 gene in Crohn's disease

[0035] 1. Experimental materials: A total of 72 paraffin specimens of endoscopic intestinal tissues were collected, including 21 cases of Crohn's disease, 23 cases of intestinal tuberculosis, 16 cases of primary intestinal lymphoma and 12 healthy controls.

[0036] 2. Experimental process: 72 paraffin specimens were collected from the pathology department of our hospital, and the paraffin specimens were cut into 10 μm thick slices, and 2-3 slices were taken. The total RNA in the sample was extracted by ALLPrep DNA / RNA FFPE Kit (QIAGEN Co.), and then the extracted RNA was reverse transcribed into cDNA by High Capacity cDNA Reverse Transcription Kit (ABI Co.). The obtained sample cDNA was processed according to the requirements of the whole genome DASL assay (Illumina Co.), followed by gene chip analysis and data processing, and the gene EphB3, which was significantly upregulated in Crohn's disease, was screened and enriched.

[0037] 3. Experimental results are as follows Figure 1 shown.

[0038] from Figure 1 It can be seen that the expression of EphB3 gene in Crohn's disease is significantly upregulated compared with its expression in intestinal tuberculosis and primary intestinal lymphoma. Therefore, by detecting the expression of EphB3 gene, the differential diagnosis between Crohn's disease and intestinal tuberculosis, or between Crohn's disease and primary intestinal lymphoma can be achieved.

[0039] Example 2

[0040] Expression of EphB3 in the intestinal mucosa of patients with Crohn's disease

[0041] 1. Another 90 paraffin specimens were collected, including 30 cases of Crohn's disease, 30 cases of intestinal tuberculosis, and 30 cases of primary intestinal lymphoma.

[0042] The steps for preparing paraffin specimens include:

[0043] 1) Collection of endoscopic intestinal tissue

[0044] After sufficient intestinal preparation, the patient will undergo colonoscopy or small enteroscopy according to the patient's actual situation, and 1-2 biopsy tissues will be taken from the lesions such as inflammation and ulcers.

[0045] 2) Fixation, dehydration and wax dipping of intestinal tissue

[0046] The intestinal tissue was fixed in 4% paraformaldehyde (Biosharp, BL539A) for 24-48 hours, and then the tissue was trimmed and placed in a dehydration box. The dehydration box containing the tissue was dehydrated using a dehydrator: 75% ethanol, 4h-85% ethanol, 2h-90% ethanol, 2h-5% ethanol, 1h-anhydrous ethanol I, 30min-anhydrous ethanol II, 30min-benzene, 10min-xylene I, 10min-xylene II, 10min-65℃ melted paraffin, 1h.

[0047] 3) Embedding and sectioning of intestinal tissue

[0048] Add molten wax to the embedding frame of the embedding machine, place the embedded tissue sections up and down, and place them in a -20℃ freezer for 1 hour; preheat the water bath and spreader, load the tissue wax block into the paraffin slicer, slowly cut the wax block containing the tissue into 4μm slices, gently spread them out in 40℃ water, use a glass slide to pick out the slices and place them on the tissue spreader, try to place the slices in the center of the slide, and dry them at 60℃ for 2 hours until the wax melts.

[0049] 2. Detection of EphB3 in paraffin sections by immunohistochemistry

[0050] The experiment includes the following steps:

[0051] 1) Dewaxing of paraffin sections: sequentially place tissue sections in xylene I, 15 min-xylene II, 15 min-xylene III, 15 min-anhydrous ethanol I, 5 min-anhydrous ethanol II, 5 min-85% ethanol, 5 min-75% ethanol, 5 min, and then wash with distilled water;

[0052] 2) Antigen retrieval: Place the dewaxed tissue sections in a retrieval box containing citric acid antigen retrieval buffer (pH 6.0) (Servicebio, G1202), heat in a microwave oven at medium heat for 8 minutes until boiling, then stop heating and heat at medium-low heat for 7 minutes; allow the tissue sections to cool naturally, then place in PBS buffer (pH 7.4) (Solarbio, P1020), shake and wash on a decolorizing shaker for 5 minutes, and repeat this step 3 times;

[0053] 3) Block endogenous peroxidase: Soak the tissue sections in 3% hydrogen peroxide solution for 25 minutes, keep away from light, then place in PBS buffer (pH 7.4), shake and wash on a decolorizing shaker for 5 minutes, and repeat this step 3 times;

[0054] 4) Serum blocking: add 3% BSA (Servicebio, G5001) to evenly cover the tissue and block at room temperature for 30 minutes;

[0055] 5) Add primary antibody: Gently shake off the blocking solution, add anti-EphB3 primary antibody (BOSTER, BA3688) prepared in proportion with PBS buffer (pH 7.4) on the tissue sections, and place the tissue sections in a humidified box and incubate at 4°C overnight;

[0056] 6) Adding secondary antibody: Place the tissue sections in PBS buffer (pH 7.4), shake and wash on a decolorizing shaker for 5 minutes, repeat this step 3 times, and after the sections are slightly dried, add the enzyme-labeled secondary antibody corresponding to the primary antibody (HRP goat anti-rabbit, Servicebio, GB23303), and then incubate at room temperature for 50 minutes;

[0057] 7) DAB color development: Place the tissue sections in PBS buffer (pH 7.4), shake and wash on a decolorizing shaker for 5 minutes, repeat this step 3 times, and after the tissue sections are slightly dried, add freshly prepared DAB color development solution (Servicebio, G1211), then control the color development time by microscopic observation, and rinse the tissue sections with tap water to stop the color development;

[0058] 8) Counterstaining of cell nuclei: Counterstain the tissue sections with hematoxylin staining solution (Servicebio, G1004) for about 3 minutes, wash with tap water, add hematoxylin differentiation solution (Servicebio, G1309), differentiate for a few seconds, wash with tap water again, then add hematoxylin bluing solution (Servicebio, G1340) to bluing, and then wash with tap water;

[0059] 9) Dehydration and sealing: Place the tissue sections in 75% ethanol, 5 min-85% ethanol, 5 min-anhydrous ethanol I, 5 min-anhydrous ethanol II, 5 min-n-butanol, 5 min-xylene I, 5 min, dehydrate until transparent, then take out the tissue sections, wait for them to dry slightly, and seal them with neutral gum;

[0060] 10) Microscopic examination: Place tissue sections under a microscope for observation;

[0061] 11) Slice scanning: Place the tissue slices in the slice slot of the pathology scanner at a time and scan them at 20 times to obtain images.

[0062] 3. Results and analysis

[0063] For each scanned image obtained from each slice, 5-10 images were randomly intercepted in the intestinal mucosal layer area, and then the images were analyzed using ImageJ software (version 2.1.0) to obtain the positive area ratio (positive expression area in the image / total tissue area in the image); then the data were statistically analyzed using IBM SPSS Statistics (version 26.0.0.2). At the same time, the accuracy, sensitivity, and specificity of differential diagnosis using EphB3 were analyzed using ROC curves.

[0064] from Figure 2 and Figure 3It can be seen that the expression level of EphB3 in the intestinal mucosa of Crohn's disease (CD) patients was significantly upregulated compared with that in the intestinal mucosa of intestinal tuberculosis (ITB) patients and primary intestinal lymphoma (PIL) patients, and the differences were statistically significant (P<0.0001).

[0065] from Figure 4 and Figure 5 It can be seen that the area under the ROC curve AUC value between Crohn's disease and intestinal tuberculosis is 0.633; the area under the ROC curve AUC value between Crohn's disease and primary intestinal lymphoma is 0.630, which proves that EphB3 as a biomarker for Crohn's disease detection has high accuracy, sensitivity and specificity.

[0066] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Application of EphB3 gene as a biomarker in the preparation of Crohn's disease detection products.

2. Application of reagents for detecting EphB3 gene expression in the preparation of Crohn's disease detection products.

3. The use according to claim 1 or 2, characterized in that: The detection product includes reagents using polymerase chain reaction technology, in situ hybridization technology, enzymatic mutation detection technology, chemical shearing mismatch technology, mass spectrometry analysis technology, gene chip technology or gene sequencing technology or a combination thereof.

4. The application according to claim 3, characterized in that: In the polymerase chain reaction technology, the polymerase chain reaction is any one of RT-PCR, immune PCR, nested PCR, fluorescent PCR, in situ PCR, membrane-bound PCR, anchored PCR, fixed PCR, in situ PCR, asymmetric PCR, long-distance PCR, parachute PCR, and gradient PCR.

5. Application of reagents for detecting the amount of EphB3 receptors in the preparation of Crohn's disease detection products.

6. The use according to claim 5, characterized in that: The detection product includes reagents using protein chip technology, ELSA or immunohistochemistry technology or a combination thereof.

7. The use according to claim 1, 2 or 5, characterized in that: The test sample of the product is an intestinal tissue sample.

8. A kit for detecting Crohn's disease, characterized in that: The kit uses polymerase chain reaction technology to detect EphB3 gene; or, The kit uses immunohistochemical analysis technology to detect EphB3 receptor.

9. The kit according to claim 8, characterized in that The kit is based on the immunohistochemical analysis technology to collect EphB3 receptor data processing method, comprising the following steps: S1, obtain intestinal tissue embedded sections, dewax, antigen retrieval, block endogenous peroxidase, block serum, and obtain pre-treated tissue sections; S2, adding anti-EphB3 primary antibody to the pre-treated tissue section for immunobinding, incubating for 10-14 hours at a temperature of 2-6° C., then adding an enzyme-labeled secondary antibody for binding to the anti-EphB3 primary antibody for immunobinding, incubating for 40-60 minutes at a temperature of 22-28° C., to obtain an immunolabeled tissue section; the marker of the enzyme-labeled secondary antibody includes horseradish peroxidase and alkaline phosphatase; S3, adding a color developing solution to the immunolabeled tissue section, counter-staining the cell nucleus, dehydrating and sealing the section, scanning to obtain a tissue image, analyzing the positive expression area ratio of the tissue image, and obtaining the expression amount data of EphB3.

10. The kit according to claim 9, characterized in that The antigen repair operation comprises: placing the dewaxed intestinal tissue embedded section in a repair box containing a citric acid antigen repair buffer solution of pH 5.8-6.5, heating for 6-10 minutes until boiling, then heating at 40-70° C. for 5-10 minutes, cooling naturally, and placing in a PBS buffer solution; The operation of blocking endogenous peroxidase comprises: soaking in a hydrogen peroxide solution with a volume fraction of 1-5% as a blocking agent for 20-30 minutes; The operation of blocking serum includes: using a BSA solution with a mass fraction of 1-5% as a blocking agent for blocking treatment for 25-35 minutes.

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