Method for constructing a mouse model of intestinal damage in sjogren's syndrome and application thereof

Abstract

The present application relates to the biomedical technology field, disclose a kind of dry syndrome intestinal damage mouse model construction method and its application.The construction method includes: (1) preparation contains Freund's complete adjuvant and 1mg / mL mouse intestinal epithelial cell antigen immunization emulsifier I and preparation contains Freund's incomplete adjuvant and 0.5mg / mL mouse intestinal epithelial cell antigen immunization emulsifier II;(2) to mouse injection the immunization emulsifier I or the immunization emulsifier II in batches, obtain the dry syndrome intestinal damage mouse model.The dry syndrome intestinal damage mouse model construction method provided by the present application only immunizes mouse by mouse intestinal epithelial cell antigen and adjuvant, obtains the mouse model that simultaneously shows dry syndrome and intestinal damage.The construction method not only has the advantages of high efficiency, fast, economic, but also is extremely easy to popularize and widely used, has good application value.

Description

A method for constructing and applying a mouse model of intestinal damage in Sjögren's syndrome Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for constructing a mouse model of intestinal damage in Sjögren's syndrome and its application. Background Technology

[0002] Primary Sjogren's syndrome (pSS) is a chronic inflammatory autoimmune disease characterized by lymphocyte proliferation and progressive damage to exocrine glands. In pSS patients, there is excessive activation of immune cells and production of autoantibodies in the salivary gland tissue. The interaction between immune cells and epithelial cells leads to impaired salivary gland secretion function.

[0003] In recent years, the diagnosis and treatment of extra-glandular manifestations of pSS have presented challenges to clinical practice because the disease has diverse clinical features, affecting the skin, lungs, kidneys, joints, muscles, peripheral nervous system, and central nervous system. However, more than a quarter of pSS patients still have overlooked systemic features, including gastrointestinal symptoms. The main gastrointestinal manifestations of pSS can occur in the esophagus, stomach, pancreas, liver, and small intestine; unfortunately, these are often not routinely assessed. Gastrointestinal involvement in pSS presents in various forms, including indigestion, constipation, diarrhea, iron deficiency anemia due to malabsorption, and celiac disease when the intestines are involved. Therefore, research into the pathogenesis and pathological mechanisms of pSS complicated by intestinal damage is crucial.

[0004] The mechanism by which pSS causes intestinal damage remains unclear. Some researchers have found an increase in nonspecific motility abnormalities in pSS patients, such as aperistalsis, triple contractions, non-peristaltic contractions, and decreased contractility. Studies on the relationship between autoimmune diseases and the gut microbiota have revealed that pSS patients have gut microbiota dysbiosis, dominated by mycobacteria, and that the gut of pSS patients contains a large number of parthenogenetic pathogens. Other studies have shown that certain gut bacteria genera are associated with elevated serum levels of pro-inflammatory cytokines and the expression of intestinal barrier damage markers (such as zonulin and FOXP3) in pSS patients. However, most published literature only describes indirect evidence obtained from clinical observations, with relatively few in-depth studies directly discussing the mechanisms of pSS-related intestinal damage, lacking a comprehensive understanding of the complete pathophysiological process of pSS-related intestinal damage. This is because there is currently a lack of animal disease models of pSS combined with intestinal damage, making it difficult to study the dynamic changes of pSS-related intestinal damage and hindering drug development. Therefore, providing a method for constructing a mouse disease model of pSS combined with intestinal damage with high success rate, short modeling time, and simple operation remains an urgent problem to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing technologies in the lack of methods for constructing animal disease models of pSS combined with intestinal damage.

[0006] To achieve the above objectives, a first aspect of the present invention provides a method for constructing a mouse model of intestinal damage in Sjögren's syndrome, the method comprising:

[0007] (1) Prepare immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen and prepare immunoemulsifier II containing Freund's incomplete adjuvant and 0.5 mg / mL of mouse intestinal epithelial cell antigen;

[0008] (2) The mouse model of intestinal damage of Sjögren's syndrome was obtained by injecting the immunoemulsifier I or the immunoemulsifier II into mice in several separate injections.

[0009] A second aspect of the present invention provides the use of a mouse model constructed according to the construction method described in the first aspect in screening drugs for the treatment and / or prevention of intestinal damage in Sjögren's syndrome.

[0010] A third aspect of the present invention provides a method for screening drugs for treating and / or preventing intestinal damage in Sjögren's syndrome, the method comprising:

[0011] (1) The test drug is administered to the mouse model of intestinal damage due to Sjögren's syndrome constructed by the construction method described in the first aspect;

[0012] (2) Analyze and evaluate the therapeutic effects of the test drugs, and select the test drugs that can significantly improve the pathological characteristics of mouse models of Sjögren's syndrome and intestinal damage.

[0013] Compared with the prior art, the present invention has at least the following advantages through the above technical solution:

[0014] (1) The method for constructing a mouse model of Sjögren's syndrome with intestinal damage provided by the present invention provides a basis for the study of the pathogenesis and intervention strategies of Sjögren's syndrome with intestinal damage.

[0015] (2) The mouse model construction method provided by the present invention has a short modeling time, high modeling rate and simple operation. The constructed mouse model not only has the pathological characteristics of Sjögren's syndrome, but also the pathological characteristics of intestinal damage, and can be used in the screening of drugs for the treatment of Sjögren's syndrome combined with intestinal damage.

[0016] (3) This invention is the first to discover that a mouse model exhibiting both Sjögren's syndrome and intestinal damage can be constructed by immunizing mice with mouse intestinal epithelial cell antigen and adjuvant. The construction method provided by this invention not only has the advantages of high efficiency, speed and economy, but is also easy to promote and widely used, and has great application value. Attached Figure Description

[0017] Figure 1 is a flowchart of the experimental process for constructing and validating the mouse model of intestinal damage in Sjögren's syndrome described in this invention.

[0018] Figure 2 shows the phenotypic verification results of the Sjögren's syndrome intestinal damage mouse model constructed in this invention.

[0019] Figure 3 shows the results of the validation of the intestinal damage phenotype in the mouse model of Sjögren's syndrome with intestinal damage constructed in this invention.

[0020] Figure 4 shows the results of the analysis of inflammatory factor levels in the colon tissue of the mouse model of intestinal damage caused by Sjögren's syndrome obtained in this invention. Detailed Implementation

[0021] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0022] As previously stated, a first aspect of the present invention provides a method for constructing a mouse model of intestinal damage due to Sjögren's syndrome, the method comprising:

[0023] (1) Prepare immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen and prepare immunoemulsifier II containing Freund's incomplete adjuvant and 0.5 mg / mL of mouse intestinal epithelial cell antigen;

[0024] (2) The mouse model of intestinal damage of Sjögren's syndrome was obtained by injecting the immunoemulsifier I or the immunoemulsifier II into mice in several separate injections.

[0025] This invention is the first to utilize mouse intestinal epithelial cell antigen plus adjuvant to immunize mice, thereby constructing a mouse model that simultaneously exhibits Sjögren's syndrome and intestinal damage. This provides a method for constructing a mouse model of Sjögren's syndrome combined with intestinal damage, laying the foundation for research on the pathogenesis and intervention strategies of Sjögren's syndrome combined with intestinal damage. Furthermore, this invention optimizes construction parameters such as antigen concentration and modeling time, resulting in a mouse model of Sjögren's syndrome combined with intestinal damage with a high success rate, stable phenotype, and a simple and easily promoted construction method.

[0026] In some embodiments, the mouse is a C57BL / 6 mouse.

[0027] Preferably, the C57BL / 6 mouse is a female C57BL / 6 mouse.

[0028] More preferably, the female C57BL / 6 mouse is an 8-week-old female C57BL / 6 mouse.

[0029] Preferably, the injection methods of the immunoemulsifier I and immunoemulsifier II are each independently selected from at least one of subcutaneous injection, intravenous injection, intraperitoneal injection, and intramuscular injection.

[0030] More preferably, the immunoemulsifier I and immunoemulsifier II are administered via subcutaneous injection. The inventors have found that, under this preferred condition, subcutaneous injection significantly stimulates a stronger immune response.

[0031] Preferably, the method for preparing the immunoemulsifier I includes: mixing Freund's complete adjuvant with 2 mg / mL of the intestinal epithelial cell antigen at a volume ratio of 1:1 to obtain immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen.

[0032] Preferably, the method for preparing the immunoemulsifier II includes: mixing Freund's incomplete adjuvant with 1 mg / mL of the intestinal epithelial cell antigen at a volume ratio of 1:1 to obtain immunoemulsifier II containing Freund's incomplete adjuvant and 0.5 mg / mL of mouse intestinal epithelial cell antigen.

[0033] Preferably, the mouse intestinal epithelial cell antigen is prepared by a method comprising the following steps: taking mouse intestinal tissue, adding neutral metalloproteinase for digestion, removing undigested mouse intestinal tissue, collecting the supernatant, and obtaining the mouse intestinal epithelial cell antigen.

[0034] Preferably, the mouse is a C57BL / 6 mouse.

[0035] More preferably, the C57BL / 6 mouse is a female C57BL / 6 mouse.

[0036] Most preferably, the female C57BL / 6 mouse is an 8-week-old female C57BL / 6 mouse.

[0037] The present invention does not have any special requirements for the method of obtaining the intestinal tissue of the mouse. It can be carried out by methods known to those skilled in the art. For example, after euthanizing the mouse by cervical dislocation, disinfect with 75% alcohol, and take 5-10 cm of intestinal tissue under sterile conditions. Peel off the capsule and connective tissue, and cut the intestine along its long axis on ice and put it into 20 mL of pre-cooled PBS. Vortex intermittently for 1-3 min to clean the intestine. Divide the intestinal tissue into 4-6 mm pieces of intestinal tissue to be digested on ice.

[0038] The present invention does not have any particular requirements for the method of removing undigested mouse intestinal tissue. Methods known to those skilled in the art can be used, such as using a 70 μm sieve to filter and remove undigested mouse intestinal tissue.

[0039] According to one specific embodiment, the method for preparing the mouse intestinal epithelial cell antigen further includes: when neutral metalloproteinase digests mouse intestinal tissue, adding DNase to lyse DNA.

[0040] In this invention, the DNA enzyme includes deoxyribonuclease I.

[0041] According to one specific embodiment, the method for preparing the mouse intestinal epithelial cell antigen further includes: terminating the digestion of mouse intestinal tissue by neutral metalloproteinases using Duchenne phosphate buffer containing 2 (v / v)% fetal bovine serum.

[0042] According to one specific embodiment, the method for preparing the mouse intestinal epithelial cell antigen further includes: after the digestion of the mouse intestinal tissue is terminated, adding erythrocyte lysis buffer for erythrocyte lysis treatment.

[0043] According to a preferred embodiment, the preparation method of the mouse intestinal epithelial cell antigen is as follows: After euthanizing the mouse by cervical dislocation, disinfect with 75% alcohol, and aseptically take 5-10 cm of intestinal tissue. Remove the capsule and connective tissue, and cut the intestine along its long axis on ice. Place the intestine in pre-cooled 20 mL of PBS and vortex intermittently for 1 min to cleanse the intestine. Divide the intestinal tissue into 5 mm segments on ice. Add 2 mL of neutral metalloproteinase for digestion for 40 min, pipetting every 10 min, and then filter using a 70 μm sieve to remove undigested tissue. Add 2 mL of DNase and continue digestion for 10 min, then add 2 mL of DuPont phosphate buffer containing 2 (v / v)% fetal bovine serum to terminate digestion. Centrifuge at 400 g for 5 min, discard the supernatant, transfer the precipitate to a 2 mL centrifuge tube, and perform lysinusoidal lysis. After lysinusoidal lysis, wash the cells, centrifuge at 400 g for 5 min, homogenize the precipitate thoroughly on ice, and incubate at 4°C at 3000 rpm. Centrifuge at rpm for 20 min, collect the supernatant, quantify the intestinal antigen concentration, and adjust the antigen concentration to 1-2 mg / mL with PBS to obtain the mouse intestinal epithelial cell antigen.

[0044] In this invention, there are no particular limitations on the method for quantifying the concentration of intestinal epithelial cell antigens. Methods known in the art can be used, such as the biuret method, Coomassie brilliant blue staining method, and BCA protein quantification method to quantify the concentration of intestinal antigens.

[0045] In this invention, the BCA protein quantification method utilizes the fact that under alkaline conditions, divalent copper ions can be reduced to monovalent copper ions by proteins. The monovalent copper ions can then interact with BCA (bicinchoninic acid), with two molecules of BCA chelating one copper ion to form a purple complex. This purple complex is water-soluble and has a characteristic absorption peak at 562 nm. Within a certain concentration range, the absorbance shows a good linear relationship with the protein content. By constructing a standard curve, the concentration of the protein to be tested can be calculated based on the absorbance at 562 nm.

[0046] In this invention, the biuret method utilizes the formation of a purple-red complex between protein and divalent copper ions in an alkaline solution. The intensity of the color is directly proportional to the protein content and independent of the protein's relative molecular mass and amino acid composition, thus it can be used to determine protein content. The determination range is generally 1-10 mg of protein.

[0047] In this invention, the Coomassie Brilliant Blue staining method utilizes the fact that Coomassie Brilliant Blue G-250 is red in its free state with a maximum absorption wavelength of 488 nm. When Coomassie Brilliant Blue G-250 binds to protein in an acidic environment, it turns cyan, causing the maximum absorption wavelength of the dye to change from 465 nm to 595 nm. Its light absorption value is directly proportional to the protein content, so it can be used for the quantitative determination of protein.

[0048] According to a specific embodiment of the present invention, the BCA protein quantification method is used to quantify the concentration of intestinal epithelial cell antigen.

[0049] In this invention, the Freund's incomplete adjuvant (FIA) is composed of liquid paraffin and lanolin in a volume ratio of 1-5:1. The volume ratio of liquid paraffin to lanolin can be adjusted as needed, and usually the volume ratio of liquid paraffin to lanolin is 2:1.

[0050] In this invention, Freund's complete adjuvant (FCA) is a water-in-oil emulsion that can very effectively induce high-titer antibody production. Freund's complete adjuvant contains cell wall components of Mycobacterium tuberculosis, which can enhance the antibody response to the antigen. The adjuvant activity derives from the sustained release of immunogen from the oil droplets and stimulates a local immune response. Generally, for the initial immunization, the Freund's complete adjuvant and antigen are emulsified (the volume ratio of Freund's complete adjuvant to antigen is 1:1). For booster immunizations, complete adjuvant, incomplete adjuvant, or no adjuvant is used. Without adjuvant, the antigen concentration increases 10-20 times. Before immunizing animals, Freund's adjuvant and antigen are mixed in a specific ratio, generally 1:1, to prepare a water-in-oil emulsion. Because the adjuvant contains sodium dodecyl sulfate (SDS), it easily emulsifies into a water-in-oil antigen emulsion complex; this emulsion must be maintained during injection into the animal.

[0051] Preferably, the fractionated injection procedure includes: on day 0, a subcutaneous injection of immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen, at a volume of 0.1 mL / mouse; on day 7, a subcutaneous injection of immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen, at a volume of 0.1 mL / mouse; and on day 14, a subcutaneous injection of immunoemulsifier II containing Freund's incomplete adjuvant and 0.5 mg / mL of mouse intestinal epithelial cell antigen, at a volume of 0.1 mL / mouse. The inventors have found that administering 0.1 mL / mouse of 1 mg / mL mouse intestinal epithelial cell antigen is appropriate; excessively high or low concentrations may induce immune tolerance.

[0052] In this invention, there are no particular limitations on the method of emulsifying the adjuvant with mouse intestinal epithelial cell antigen. Methods known in the art can be used, such as grinding, syringe mixing, and ultrasound to emulsify the adjuvant with mouse intestinal epithelial cell antigen.

[0053] In this invention, the grinding method involves first heating the adjuvant and placing an appropriate amount into a sterile glass mortar. After cooling, an equal volume of antigen solution is slowly added dropwise while grinding in the same direction. The antigen is added slowly. After all the antigen has been added, grinding continues for a period of time until a milky white, viscous water-in-oil emulsion is formed. This method is suitable for preparing large quantities of adjuvant antigen, but its disadvantage is that a large amount of emulsion adheres to the mortar wall, resulting in significant antigen loss.

[0054] In this invention, the syringe mixing method involves drawing equal volumes of Freund's adjuvant and antigen solution into two separate syringes, connected by a thin rubber tube. Care is taken to expel all air, and the syringes are then alternately pushed until a viscous emulsion is formed. The advantages of this syringe mixing method are that it allows for aseptic operation, minimizes antigen loss, and is suitable for preparing small quantities of antigen emulsions. However, it is difficult to achieve complete emulsification; some antigens are difficult to move using a plastic syringe, while glass syringes may leak. The prepared emulsion must be tested before use. The testing method involves dropping the emulsion into cold water. If it remains intact and floats on the surface as a drop, the emulsification is complete, and it is a qualified water-in-oil emulsion suitable for subsequent experimental research.

[0055] In this invention, the ultrasonic method uses an ultrasonic disruptor to emulsify the adjuvant and antigen. During the operation, it is essential to control the ultrasonic frequency and time, as ultrasound can easily excite some free radicals, which may cause unknown damage to the antigen.

[0056] According to one specific embodiment of the present invention, the method for emulsifying the adjuvant and antigen is a syringe mixing method.

[0057] Preferably, the injection site is selected from at least one of the mouse's groin, abdomen, and back of the neck.

[0058] Preferably, the injection site is the back of the neck.

[0059] According to one specific embodiment of the present invention, an emulsion is prepared by homogenizing intestinal epithelial cells of C57BL / 6 mice with Freund's adjuvant. This emulsion is then injected subcutaneously at multiple sites into the neck and back of allotype mice to mediate an inflammatory immune response, inducing lymphocyte infiltration and interfering with the immune system. Multiple subcutaneous injections can generate a strong immune response. The antigen is injected in three doses: the antigen concentration is adjusted using Freund's complete adjuvant on days 0 and 7, and a parallel operation is performed using Freund's incomplete adjuvant on day 14 to enhance the local immune response.

[0060] Furthermore, the number of injections and intervals of the antigen described in this invention are appropriate. Frequent injections may cause the mice to experience a decline in physiological and mental state, making them more prone to death.

[0061] According to a specific embodiment of the present invention, the present invention also provides a method for validating the mouse model of Sjögren's syndrome combined with intestinal damage, the method comprising the following steps:

[0062] (1) The mouse model of Sjögren's syndrome combined with intestinal damage was constructed using the construction method described in the first aspect, and control mice without treatment were set up;

[0063] (2) After modeling, the status of the mouse model of Sjögren's syndrome combined with intestinal damage and the control mice were observed. The mouse weight, water intake, saliva volume, serum anti-SSA antibody level, serum anti-SSB antibody level, serum motilin level, colon length, intestinal and submandibular gland histopathology and colon tissue inflammatory factor level were detected.

[0064] In this invention, the observation of mouse condition refers to observing whether the mice scratch their lips or lick their paws around the third week after modeling.

[0065] In this invention, the method for detecting saliva volume includes: before measurement, mice are intraperitoneally injected with ready-to-use tribromoethanol solution at a dose of 0.2 mL / 10g body weight. Anesthesia is achieved when breathing is stable, corneal reflex is absent, and limb muscles are relaxed. After complete anesthesia, the mouse is placed in a slightly tilted position with its head lowered, and a warming pad can be used to maintain temperature. A 0.025 mg / mL pilocarpine solution is injected intraperitoneally at a dose of 0.1 mL / 20g body weight. Five minutes later, a 100-200 mg cotton ball is inserted into the mouse's mouth. After 10 minutes, the cotton ball is removed, and the weight difference before and after is measured to determine the mouse's saliva weight. To reduce errors caused by saliva evaporation, after removing the cotton ball from the mouse's mouth, it can be placed in a 1.5 mL EP tube. During the experiment, the EP tube is placed on ice.

[0066] In this invention, the method for histopathological examination of the intestine and submandibular gland tissue includes: taking the intestine and submandibular gland of mice at the 10th week of modeling, and performing histopathological examination of the intestine and submandibular gland using H&E staining.

[0067] In this invention, the operation of the H&E staining method includes: the tissue should first be soaked in fixative for 24 hours, and then rinsed, dehydrated and cleared, impregnated with paraffin, embedded, paraffin sectioned, and H&E stained in sequence.

[0068] Preferably, the fixative is 4% paraformaldehyde.

[0069] In this invention, the method for detecting the level of inflammatory factors in colon tissue includes: ultrasonically disrupting a colon sample, centrifuging it at 8000-10000 rpm for 8-10 min at 4°C, and collecting the supernatant. The total protein content of the supernatant is measured according to the instructions of the BCA protein concentration kit to ensure a consistent total protein content, which serves as background correction for inflammatory factor levels. The levels of inflammatory cytokines IL-6, IL-1β, TNF-α, and MPO in the supernatant are then determined using ELISA technology.

[0070] As previously stated, a second aspect of the present invention provides the use of a mouse model constructed according to the construction method described in the first aspect in screening drugs for the treatment and / or prevention of intestinal damage in Sjögren's syndrome.

[0071] As previously described, a third aspect of the present invention provides a method for screening drugs for treating and / or preventing intestinal damage in Sjögren's syndrome, the method comprising:

[0072] (1) The test drug is administered to the mouse model of intestinal damage due to Sjögren's syndrome constructed by the construction method described in the first aspect;

[0073] (2) Analyze and evaluate the therapeutic effects of the test drugs, and select the test drugs that can significantly improve the pathological characteristics of mouse models of Sjögren's syndrome and intestinal damage.

[0074] The present invention will be described in detail below through examples. Unless otherwise specified, all reagents used were purchased from reputable chemical or biological reagent suppliers and were of analytical grade.

[0075] The experimental flowchart for the construction and validation of the mouse model of intestinal damage due to Sjögren's syndrome described in this invention is shown in Figure 1.

[0076] 1. Experimental materials

[0077] C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. They were 6 weeks old, female, and housed in the SPF-grade laboratory of the Laboratory Animal Department of Capital Medical University at a temperature of 23±1℃ and humidity of 50%-60%, with no dietary restrictions. The animals underwent a one-week quarantine and inspection process before being transferred to the laboratory, and after passing the tests, they were placed in the aforementioned laboratory for another week of acclimatization. This experimental protocol for these mice has been approved by the Laboratory Animal Welfare and Ethics Committee of Capital Medical University.

[0078] 2. Medicines and reagents

[0079] Freund's Complete Adjuvant (FCA): Purchased from Sigma-Aldrich, USA, catalog number F5881.

[0080] Freund's incomplete adjuvant (FIA): purchased from Sigma-Aldrich, USA, catalog number F5506.

[0081] Phosphate-buffered saline (PBS): purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number P1020.

[0082] Neutral metalloproteinase: purchased from Corning, Inc., USA, catalog number 354235.

[0083] Ready-to-use tribromoethanol solution: Purchased from Nanjing Aibei Biotechnology Co., Ltd., product number M2920, is a ready-to-use sterile solution containing 1.25% (v / v) tribromoethanol (Avorin), tert-amyl alcohol, and 0.9% physiological saline. The final concentration of tribromoethanol is 20 mg / mL. The injection dose for mice was calculated at 0.2 mL / 10 g body weight.

[0084] Pilocarpine: Purchased from Selleck Chemicals, USA, catalog number S4231, white powder. Weigh 10 mg of pilocarpine and dissolve it in 10 mL of double-distilled water to prepare 1 mg / mL pilocarpine solution. When using, dilute to the required concentration (0.025 mg / mL pilocarpine solution). The dosage is 0.1 mL intraperitoneally injected into mice weighing 20 g.

[0085] BCA protein concentration assay kit: purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number PC0020.

[0086] Red blood cell lysis buffer: purchased from Beijing Solarbio Science & Technology Co., Ltd., product number R1010.

[0087] Hematoxylin and eosin (H&E) staining kit: purchased from Beijing Solarbio Science & Technology Co., Ltd., product number G1120.

[0088] Carmine: Purchased from Sigma-Aldrich, USA, product number C1022.

[0089] Methylcellulose: purchased from Sigma-Aldrich, USA, catalog number M0512.

[0090] Mouse serum anti-SSA antibody ELISA kit: purchased from Alpha Diagnostic International, USA, catalog number 5710.

[0091] Mouse serum anti-SSB antibody ELISA kit: purchased from Alpha Diagnostic International, USA, catalog number 5810.

[0092] Mouse serum motilin ELISA kit: purchased from Shanghai Enzyme-Link Biotechnology Co., Ltd., catalog number ml201829.

[0093] The optical microscope was purchased from ZEISS in Germany, model Axio Lab.A1.

[0094] Deoxyribonuclease I: purchased from Stemcell Technologies, catalog number 07469.

[0095] 70 μm sieve: purchased from Corning, USA, part number 352350.

[0096] 100 μm sieve: purchased from Corning, USA, part number 431752.

[0097] Duchenne phosphate buffer containing 2% fetal bovine serum: purchased from Stemcell Technologies, catalog number 07905.

[0098] The IL-6 ELISA kit was purchased from R&D Systems, catalog number M6000B-1.

[0099] The IL-1β ELISA kit was purchased from R&D Systems, catalog number MLB00C-1.

[0100] The TNF-α ELISA kit was purchased from R&D Systems, catalog number MTA00B-1.

[0101] The MPO ELISA kit was purchased from Abcam, catalog number ab155458.

[0102] Collagenase I was purchased from Thermo Fisher Scientific, Inc., USA, catalog number 17100017.

[0103] The method for preparing a 1% hydrochloric acid-ethanol solution is as follows: Measure 1 mL of concentrated hydrochloric acid with a pipette and 99 mL of 70% ethanol with a graduated cylinder. In a fume hood, slowly add the concentrated hydrochloric acid to the 70% ethanol while gently stirring with a glass rod to ensure thorough mixing.

[0104] The preparation method for a 1% ammonia blue solution is as follows: Measure 2 mL of concentrated ammonia using a graduated cylinder. Add approximately 150 mL of distilled water to a beaker. In a fume hood, slowly add the measured concentrated ammonia to the distilled water while gently stirring. Finally, rinse the graduated cylinder with distilled water and combine the liquids in a container, bringing the final volume to 200 mL, and stir thoroughly.

[0105] Methods for detecting serum anti-SSA antibody levels:

[0106] The serum anti-SSA antibody level was detected using a mouse serum anti-SSA antibody ELISA kit. The specific steps are as follows:

[0107] (1) Sample collection and preservation: Whole blood samples were placed at room temperature for 2 hours and then centrifuged at 12000g for 15 minutes at 4℃. The supernatant was collected, aliquoted, and stored at -20℃ to avoid repeated freeze-thaw cycles. The thawed serum samples were centrifuged again at 12000g for 15 minutes at 4℃ before testing.

[0108] (2) Reagent preparation: Dilute the plate washing solution stock solution 100 times (add 10 mL of plate washing solution stock solution to 990 mL of distilled water) as the plate washing solution working solution and store at room temperature; dilute the mouse serum sample 20 times (add 10 mL of mouse serum sample to 190 mL of distilled water) as the diluted mouse serum sample and store at 4℃;

[0109] (3) Washing the plate: Add 250 μL of washing solution to each well of the 96-well plate containing the solid primary antibody, do not blow or agitate, let stand for 5 minutes, and pat dry on paper;

[0110] (4) First incubation: Add 100 μL of deionized water (blank control), standard and diluted mouse serum sample to each well after step (3), gently blow and let stand at room temperature for 60 min, and wash the plate 4 times.

[0111] (5) Second incubation: Add 100 μL of enzyme-labeled reagent (secondary antibody) to each well, let stand for 30 min, and wash the plate 5 times;

[0112] (6) Substrate incubation: In the dark, add 100 μL of TMB to each well, let stand for 15 min, and the system will turn blue;

[0113] (7) Termination: Add 100 μL of stop solution to each well, gently blow and the system turns yellow;

[0114] (8) Color development: After shaking and mixing, measure the OD value at a wavelength of 450 nm using an ELISA reader;

[0115] (9) Calculation: Construct a standard curve, calculate the anti-SSA antibody titer corresponding to the sample, and perform statistical analysis. Precautions: ① The kit should be equilibrated at room temperature for at least 30 minutes after being taken out of the refrigerated environment before use. If the enzyme-labeled plate is not used up after opening, the strips should be stored in a sealed bag; ② The concentrated washing solution may crystallize. When diluting, it can be warmed in a water bath to help dissolve. This will not affect the washing effect.

[0116] ③ A pipette should be used for each step of sample addition, and its accuracy should be calibrated to avoid experimental errors. The sample addition time should ideally be controlled within 5 minutes. If there are many samples, it is recommended to use a multi-channel pipette. ④ A standard curve should be prepared at the same time as each measurement, and it is best to make duplicates.

[0117] Methods for detecting serum anti-SSB antibody levels

[0118] The serum anti-SSB antibody level was detected using a mouse serum anti-SSB antibody ELISA kit, following the same procedure as the serum anti-SSA antibody level detection procedure.

[0119] Serum motilin detection method:

[0120] The serum motilin level was detected using a mouse serum motilin ELISA kit. The specific steps are as follows:

[0121] (1) Sample collection and preservation: Whole blood samples were placed at room temperature for 2 hours, then centrifuged at 12000g for 15 min at 4℃. The supernatant was collected, aliquoted, and stored at -20℃ to avoid repeated freeze-thaw cycles. The thawed supernatant samples (mouse serum samples) were centrifuged again at 12000g for 15 min at 4℃ before testing.

[0122] (2) Addition of standards: Set up standard wells and add 50 μL of each of the following standards: 400 pg / mL, 200 pg / mL, 100 pg / mL, 50 pg / mL, 25 pg / mL and 0 pg / mL respectively.

[0123] (3) Sample addition: Set up blank wells (blank control wells do not contain supernatant sample and enzyme labeling reagent, and the other steps are the same) and sample wells. First, add 40 μL of sample diluent to the sample wells of the enzyme-labeled plate, and then add 10 μL of mouse serum sample to be tested (the supernatant sample to be tested is finally diluted 5 times). Add the supernatant sample to the bottom of the well of the enzyme-labeled plate, trying not to touch the well wall, and gently shake to mix.

[0124] (4) Incubation: After sealing with sealing film, incubate at 37°C for 30 minutes;

[0125] (5) Solution preparation: Dilute the 30-fold concentrated washing solution with distilled water 30 times to prepare the washing solution for later use;

[0126] (6) Washing: Remove the sealing film, discard the liquid, spin dry, fill each well with washing liquid, let stand for 30 seconds and then discard, repeat this 5 times, and pat dry with absorbent paper;

[0127] (7) Add enzyme: Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells;

[0128] (8) Incubation: The procedure is the same as (4);

[0129] (9) Washing: Same as (6);

[0130] (10) Color development: Add 50 μL of color developer A to each well, then add 50 μL of color developer B, gently shake to mix, and develop color at 37°C in the dark for 15 minutes.

[0131] (11) Termination: Add 50 μL of stop solution to each well to terminate the reaction;

[0132] (12) Measurement: Zero the blank well and use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm in sequence. The measurement should be performed within 15 minutes after adding the stop solution.

[0133] HE staining of paraffin sections

[0134] (1) Preparation and sectioning of paraffin-embedded specimens

[0135] ① Fixation: Immediately after specimen collection, immerse the specimen in 4% paraformaldehyde for 24 hours. The volume of 4% paraformaldehyde should be 20 times the volume of the tissue. After fixation, remove the specimen with forceps and place the trimmed tissue and corresponding label into a dehydration box.

[0136] ② Dehydration: Take out the tissue and place it in 70 (v / v)% ethanol, 80 (v / v)% ethanol, 90 (v / v)% ethanol, 95 (v / v)% ethanol, 95 (v / v)% ethanol and 100 (v / v)% ethanol in sequence for 20 min each time.

[0137] ③ Transparency: After dehydration, the specimen is placed in xylene solution twice, each time for 20 minutes;

[0138] ④ Paraffin permeation: Prepare a mixture of xylene and paraffin at a volume ratio of 1:1. Immerse the cleared tissue in the mixture for 20 minutes, then immerse it in paraffin twice, each time for 1 hour. All the above steps should be performed in a constant temperature oven at 58℃.

[0139] ⑤ Embedding: Remove the wax cup from the 60℃ constant temperature incubator, pour in 5mL of paraffin wax to melt it, and heat the wax mold over an alcohol lamp before placing it flat. Use tweezers to pick up the tissue and place it in the prepared wax mold with the cut surface facing down. Then add the embedding box and gently pour the melted paraffin wax into the box for tissue paraffin embedding.

[0140] ⑥ Sectioning: Place the embedded paraffin tissue block in the microtome, fix it with the stage, and then use the microtome blade to cut the paraffin tissue block into 3 μm sections;

[0141] ⑦ Spreading: Use a blade to cut the wax strip, and tweezers to pick up the slides and place them in the ethanol solution, spreading each slide out. During the spreading process, use tweezers to separate any slides that are connected, and use a glass slide to make the slides spread out more completely;

[0142] ⑧ Slide mounting: After the slide has fully expanded, it can be transferred to warm water and placed for 1 minute. Then take it out and take another clean glass slide. Attach the retrieved slide to 1 / 3 of the slide and attach a label to the other end of the glass slide.

[0143] ⑨ Baking the slides: Place the glass slides in a 60℃ oven and bake for 2 hours, then place them in a 37℃ incubator to fix for 12 hours. After the operation is completed, place the slides on a slide rack.

[0144] (2) HE staining

[0145] ① Dewaxing: Place the slices in xylene solution overnight to dewax.

[0146] ② Hydration: After dewaxing, the sections are placed in 100 (v / v)% ethanol, 95 (v / v)% ethanol, 75 (v / v)% ethanol and 50 (v / v)% ethanol solutions in sequence. After each soaking for 5 minutes, the sections are taken out and rinsed with running water.

[0147] ③ Hematoxylin staining: After rinsing with running water, immerse the sections in hematoxylin solution for 3 min;

[0148] ④ Hydrochloric acid-alcohol differentiation: Separate colors with 1% hydrochloric acid-alcohol solution for 3 seconds, then rinse with running water;

[0149] ⑤ Ammonia blueing: Immerse in a 1% ammonia blueing solution for 10 seconds, then rinse with running water;

[0150] ⑥ Eosin staining: Immerse in eosin solution for 2 min, then rinse with running water;

[0151] ⑦ Dehydration: Immerse in 50 (v / v)% ethanol, 75 (v / v)% ethanol, 95 (v / v)% ethanol and 100 (v / v)% ethanol solutions in sequence, soaking for 5 minutes each time;

[0152] ⑧ Transparency: Place the specimen rack in xylene and soak for 10 minutes, then replace with new xylene and soak for another 10 minutes;

[0153] ⑨ Mounting: Mount the sections with neutral transparent resin, place them horizontally, and let them air dry to obtain HE stained sections;

[0154] ⑩ Observation: HE-stained sections were observed using an optical microscope.

[0155] Example 1

[0156] Construction of a mouse model of intestinal damage in Sjögren's syndrome

[0157] (1) Preparation of antigen

[0158] a. Preparation of mouse intestinal epithelial cell antigen: C57BL / 6 mice were euthanized by cervical dislocation, disinfected with 75% (v / v)% alcohol, and under aseptic conditions, 8 cm of intestinal tissue was harvested. The capsule and connective tissue were peeled off, and the intestine was dissected along its long axis on ice and placed into a 50 mL centrifuge tube (pre-filled with 20 mL of cold PBS). The tube was intermittently vortexed for 1 min to cleanse the intestine. Then, on ice, the intestinal tissue was divided into segments of about 5 mm. 2 mL of neutral metalloproteinase was added for digestion for 40 min, with the tube being pipetted every 10 min to ensure even digestion. The mixture was filtered through a 70 μm sieve to remove undigested tissue, yielding a suspension. 2 mL of deoxyribonuclease I was added, and digestion continued for 10 min. Then, 2 mL of DuPont phosphate buffer containing 2% (v / v)% fetal bovine serum was added to terminate the digestion. The mixture was centrifuged at 400 g for 5 min, the supernatant was discarded, and the precipitate was transferred to a 2 mL centrifuge tube. Four times the cell volume of erythrocyte lysis buffer was added for erythrocyte lysis. After red blood cell lysis, wash the cells, centrifuge at 400g for 5 min, homogenize the precipitate thoroughly in an ice bath, centrifuge at 3000 rpm for 20 min at 4℃, collect the supernatant, and quantify the intestinal antigen concentration using a BCA protein concentration assay kit.

[0159] The concentration of mouse intestinal epithelial cell antigen was adjusted to 2 mg / mL using PBS, and an equal volume of Freund's complete adjuvant was added. The concentration of mouse intestinal epithelial cell antigen was diluted to 1 mg / mL by repeated pipetting until the two solutions were miscible and milky white. This prepared 1 mg / mL mouse intestinal epithelial cell antigen prepared with Freund's complete adjuvant for injection. The concentration of mouse intestinal epithelial cell antigen was adjusted to 1 mg / mL using PBS, and an equal volume of Freund's incomplete adjuvant was added. The concentration of mouse intestinal epithelial cell antigen was diluted to 0.5 mg / mL by repeated pipetting until the two solutions were miscible and milky white. This prepared 0.5 mg / mL mouse intestinal epithelial cell antigen prepared with Freund's incomplete adjuvant for injection.

[0160] b. Preparation of mouse submandibular gland antigen

[0161] ① Eight-week-old female C57BL / 6 mice were sacrificed by cervical dislocation, disinfected with 75 (v / v)% alcohol, and the bilateral submandibular glands were removed under aseptic conditions;

[0162] ② After peeling off the capsule and connective tissue, wash with PBS and place in a sterile penicillin vial. Add PBS to the sterile penicillin vial according to the amount of 0.5 mL added to each submandibular gland.

[0163] ③ Cut the submandibular gland tissue in the bottle into small pieces and transfer it to a 2 mL cell cryopreservation tube. Add 2 mL of 1.25 mg / mL collagenase I and digest at 37°C for 1 h. Pipe the tube every 15 min to make the digestion more uniform.

[0164] ④ Use a 100 μm sieve to filter out undigested tissue and obtain a suspension;

[0165] ⑤ Homogenize the suspension thoroughly in an ice bath, centrifuge at 3000g at 4℃ for 20 min, and collect the supernatant;

[0166] ⑥ The protein concentration in the supernatant was quantified using a BCA protein concentration assay kit. The protein concentration was adjusted to 4 mg / mL with PBS, and an equal volume of Freund's complete adjuvant was added. The mixture was repeatedly pipetted until the two solutions were miscible and milky white, thus obtaining 2 mg / mL mouse submandibular gland antigen prepared with Freund's complete adjuvant for injection. The protein concentration was adjusted to 2 mg / mL with PBS, and an equal volume of Freund's incomplete adjuvant was added. The mixture was repeatedly pipetted until the two solutions were miscible and milky white, thus obtaining 1 mg / mL mouse submandibular gland antigen prepared with Freund's incomplete adjuvant for injection.

[0167] (2) Eight-week-old female C57BL / 6 mice were randomly divided into four groups to ensure that the number, weight, and condition of mice in each group were as similar as possible. The hair on the back of the neck of the mice was shaved off with scissors or an electric razor in preparation for antigen injection. The treatment of each mouse is as follows:

[0168] ① Intestinal antigen immunization group (immunized with Intestinal Epithelial Cell Extracts, IEC group) (n=4), also known as the subcutaneous injection of intestinal protein extract group mice: On day 0 and day 7, mice were injected subcutaneously at multiple points on the back of the neck with 1 mg / mL mouse intestinal epithelial cell antigen prepared with Freund's complete adjuvant, with an injection volume of 0.1 mL / mouse; on day 14, mice were injected with 0.5 mg / mL mouse intestinal epithelial cell antigen prepared with Freund's incomplete adjuvant, with an injection volume of 0.1 mL / mouse.

[0169] ② Submandibular gland protein immunization group (immunized with Salivary Gland extracts, SG group) (n=4): On day 0 and day 7, mice were injected subcutaneously at multiple points on the back of the neck with 2 mg / mL mouse submandibular gland antigen prepared with Freund's complete adjuvant, with an injection volume of 0.1 mL / mouse; on day 14, mice were injected with 1 mg / mL mouse submandibular gland antigen prepared with Freund's incomplete adjuvant, with an injection volume of 0.1 mL / mouse.

[0170] ③ PBS control group (PBS group) (n=4): The same amount of PBS was injected subcutaneously;

[0171] ④ Adjuvant control group (n=4): The same amount of adjuvant was administered subcutaneously.

[0172] (3) Detection indicators after modeling: Screening mice that have successfully modeled the mice. The detection indicators include: observation of mouse status, recording of mouse weight, detection of water intake, detection of saliva volume, detection of serum anti-SSA antibody level, detection of serum anti-SSB antibody level, detection of serum motilin, analysis of colon length, pathological examination of intestinal and submandibular gland tissues, and analysis of colon tissue inflammatory factor levels.

[0173] Mouse condition observation: Around the 3rd week after modeling, observe whether the mice exhibit scratching of their lips or licking of their paws.

[0174] Mouse weight record: The weight of mice was recorded at week 10 after modeling.

[0175] The method for detecting saliva volume was as follows: Before measurement, mice were intraperitoneally injected with ready-to-use tribromoethanol solution at a dose of 0.2 mL / 10 g body weight. Anesthesia criteria included stable breathing, loss of corneal reflex, and relaxation of limb muscles. After complete anesthesia, the mice were positioned with their heads lowered and slightly tilted, and a warming pad was used to maintain temperature. Pilocarpine solution (0.025 mg / mL) was injected intraperitoneally at a dose of 0.1 mL / 20 g body weight. Five minutes later, a 150 mg cotton ball was inserted into the mouse's mouth. After 10 minutes, the cotton ball was removed, and the weight difference before and after removal was measured to determine the mouse's saliva weight. To reduce errors caused by saliva evaporation, the cotton ball was placed in a 1.5 mL EP tube after being removed from the mouse's mouth. The EP tube was kept on ice during the experiment.

[0176] Colon length analysis method: After euthanasia, the mice were dissected, the colon (from the ileocecal junction to the end of the rectum) was separated, the contents were removed and rinsed with PBS; the colon was laid flat on filter paper and the total length was measured with a ruler (avoiding stretching).

[0177] The histopathological examination methods for intestinal and submandibular gland tissues were as follows: Mice were sacrificed at week 10 of modeling, and intestinal and submandibular gland tissues were collected for pathological examination using the H&E staining method. The severity of lymphocyte infiltration in the submandibular gland tissue was used to score the H&E staining pathology of the submandibular glands in mice immunized with intestinal antigen, mice immunized with submandibular gland protein, mice in the PBS control group, and mice in the adjuvant control group. The scoring criteria were: per 4 mm... 2 More than 50 lymphocytes were defined as lymphocytic foci. The five scores were 0 (no lymphocytic infiltration), 1 (few scattered lymphocytic infiltrations), 2 (moderate lymphocytic infiltrations but not foci), 3 (1 lymphocytic infiltration foci), and 4 (more than 1 lymphocytic infiltration foci).

[0178] The method for analyzing the levels of inflammatory factors in colon tissue was as follows: An appropriate amount of colon sample was weighed, added to physiological saline, and then sonicated (ultrasound power set to 250 W, operating for 5 seconds, with 7-second intervals, for a total of 3 minutes). The sonicated sample was centrifuged at 10,000 r / min for 10 minutes at 4℃, and the supernatant was collected. The total protein content of the supernatant was measured according to the instructions of the BCA protein concentration kit, serving as background correction for inflammatory factor levels. The levels of the inflammatory cytokine IL-6 in the supernatant were measured according to the instructions of the IL-6 ELISA kit. The levels of the inflammatory cytokine IL-1β in the supernatant were measured according to the instructions of the IL-1β ELISA kit. The levels of the inflammatory cytokine TNF-α in the supernatant were measured according to the instructions of the TNF-α ELISA kit. The levels of the inflammatory cytokine MPO in the supernatant were measured according to the instructions of the MPO ELISA kit.

[0179] (4) Experimental results

[0180] Observations revealed that, compared with the control groups (PBS control group and adjuvant control group), mice in the subcutaneous injection group of intestinal protein extract exhibited scratching of their lips and lethargy.

[0181] Figure 2 shows the phenotypic verification results of the Sjögren's syndrome intestinal damage mouse model constructed in this invention.

[0182] The saliva volume of mice immunized with intestinal antigen, mice immunized with submandibular gland protein, mice in the PBS control group, and mice in the adjuvant control group is shown in Figure 2(A). As can be seen from Figure 2(A), the saliva volume of mice immunized with intestinal antigen and mice immunized with submandibular gland protein was significantly reduced.

[0183] The water intake of mice in the intestinal antigen immunization group, the submandibular gland protein immunization group, the PBS control group, and the adjuvant control group is shown in Figure 2 (B). As can be seen from Figure 2 (B), the water intake of mice in the subcutaneous injection of intestinal protein extract group increased significantly.

[0184] The results of detecting the levels of anti-SSA autoantibodies in the serum of mice immunized with intestinal antigen, mice immunized with submandibular gland protein, mice in the PBS control group, and mice in the adjuvant control group are shown in Figure 2 (C). As can be seen from Figure 2 (C), the levels of anti-SSA autoantibodies in the serum of mice immunized with intestinal antigen and mice immunized with submandibular gland protein were significantly increased.

[0185] The results of detecting the level of anti-SSB autoantibodies in the serum of mice immunized with intestinal antigen, mice immunized with submandibular gland protein, mice in the PBS control group, and mice in the adjuvant control group are shown in Figure 2(D). As can be seen from Figure 2(D), the level of anti-SSB autoantibodies in the serum of mice immunized with intestinal antigen and mice immunized with submandibular gland protein was significantly increased.

[0186] At week 10 of modeling, the HE staining results of the submandibular glands of mice in the intestinal antigen immunization group, the submandibular gland protein immunization group, the PBS control group, and the adjuvant control group are shown in Figure 2(E). As can be seen from Figure 2(E), at week 10 of modeling, the submandibular glands of mice in the intestinal antigen immunization group and the submandibular gland protein immunization group showed severe lymphocyte infiltration.

[0187] The severity of lymphocyte infiltration in the submandibular gland tissue was assessed by HE staining of the submandibular glands in mice immunized with intestinal antigen, mice immunized with submandibular gland protein, mice in the PBS control group, and mice in the adjuvant control group. The results are shown in Figure 2(F). As can be seen from Figure 2(F), the histological scores of the submandibular glands in mice immunized with intestinal antigen and mice immunized with submandibular gland protein were significantly increased at week 10 of modeling.

[0188] Figure 3 shows the results of the validation of the intestinal damage phenotype in the mouse model of Sjögren's syndrome with intestinal damage constructed in this invention.

[0189] The recorded body weights of mice in the intestinal antigen immunization group, the submandibular gland protein immunization group, the PBS control group, and the adjuvant control group are shown in Figure 3(A). As can be seen from Figure 3(A), compared with the control group, the body weight of mice in the subcutaneous injection group of intestinal protein extract was significantly reduced.

[0190] The colon lengths of mice immunized with intestinal antigens, mice immunized with submandibular gland protein, mice in the PBS control group, and mice in the adjuvant control group are shown in Figure 3(B). As can be seen from Figure 3(B), compared with the control group, the colons of mice in the subcutaneous injection group of intestinal protein extract were significantly shorter.

[0191] The motilin levels in mice immunized with intestinal antigen, mice immunized with submandibular gland protein, mice in the PBS control group, and mice in the adjuvant control group are shown in Figure 3(C). As can be seen from Figure 3(C), compared with the control group, the serum motilin level in mice subcutaneously injected with intestinal protein extract was significantly reduced.

[0192] The HE staining results of intestinal tissue are shown in Figure 3(D). As can be seen from Figure 3(D), compared with the control group, under light microscopy, the colonic mucosal epithelial structure of the mice injected subcutaneously with intestinal protein extract was damaged, the glands in the lamina propria were disordered or even disappeared, and inflammatory cells infiltrated into the submucosa. No pathological damage was observed in the colonic tissue of the control group mice, the mucosa and muscle layer were normal, the glandular structure was orderly arranged, and no inflammatory cell infiltration was observed.

[0193] Figure 4 shows the results of the analysis of inflammatory factor levels in the colon tissue of the mouse model of intestinal damage caused by Sjögren's syndrome obtained in this invention.

[0194] The level of the inflammatory cytokine TNF-α was detected using ELISA technology. The results are shown in Figure 4(A). As can be seen from Figure 4(A), compared with the control group, subcutaneous immunization with intestinal protein extract can increase the content of the inflammatory cytokine TNF-α in colon tissue.

[0195] The level of the inflammatory cytokine IL-6 was detected using ELISA technology. The results are shown in Figure 4(B). As can be seen from Figure 4(B), compared with the control group, subcutaneous immunization with intestinal protein extract can increase the content of the inflammatory cytokine IL-6 in colon tissue.

[0196] The level of the inflammatory cytokine IL-1β was detected using ELISA technology. The results are shown in Figure 4(C). As can be seen from Figure 4(C), compared with the control group, subcutaneous immunization with intestinal protein extract can increase the content of the inflammatory cytokine IL-1β in colon tissue.

[0197] The MPO level was detected using ELISA technology, and the results are shown in Figure 4(D). As can be seen from Figure 4(D), compared with the control group, subcutaneous immunization with intestinal protein extract can increase the content of inflammatory cytokine MPO in colon tissue.

[0198] In summary, the results of decreased saliva secretion, polydipsia, weight loss, colon shortening, and inflammatory cell infiltration in the colonic tissue of mice immunized with intestinal antigen all indicate that the mouse model constructed in this invention is a mouse model of Sjögren's syndrome combined with intestinal damage. Furthermore, the results show that the optimal concentration of 0.1 mL / mouse is 1 mg / mL of mouse intestinal epithelial cell antigen, while excessively high or low concentrations can easily induce immune tolerance.

[0199] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.

Claims

1. A method for constructing a mouse model of intestinal damage due to Sjögren's syndrome, characterized in that, The method includes: (1) preparing immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen, and preparing immunoemulsifier II containing Freund's incomplete adjuvant and 0.5 mg / mL of mouse intestinal epithelial cell antigen; (2) injecting the immunoemulsifier I and the immunoemulsifier II into mice in fractional injections to obtain the mouse model of Sjögren's syndrome intestinal damage; the fractional injection operation includes: on day 0, subcutaneously injecting immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen, with an injection volume of 0.1 mL / mouse; on day 7, subcutaneously injecting immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen, with an injection volume of 0.1 mL / mouse; on day 14, subcutaneously injecting immunoemulsifier II containing Freund's incomplete adjuvant and 0.5 mg / mL of mouse intestinal epithelial cell antigen, with an injection volume of 0.1 mL / mouse.

2. The construction method according to claim 1, characterized in that, The mice in question were C57BL / 6 mice.

3. The construction method according to claim 1, characterized in that, The preparation method of the immunoemulsifier I includes: mixing Freund's complete adjuvant with 2 mg / mL of the intestinal epithelial cell antigen at a volume ratio of 1:1 to obtain immunoemulsifier I containing Freund's complete adjuvant and 1 mg / mL of mouse intestinal epithelial cell antigen.

4. The construction method according to claim 1, characterized in that, The method for preparing the immunoemulsifier II includes: mixing Freund's incomplete adjuvant with 1 mg / mL of the intestinal epithelial cell antigen at a volume ratio of 1:1 to obtain immunoemulsifier II containing Freund's incomplete adjuvant and 0.5 mg / mL of mouse intestinal epithelial cell antigen.

5. The construction method according to claim 3 or 4, characterized in that, The mouse intestinal epithelial cell antigen is prepared by a method including the following steps: taking mouse intestinal tissue, adding neutral metalloproteinase for digestion, removing undigested mouse intestinal tissue, collecting the supernatant, and obtaining the mouse intestinal epithelial cell antigen.

6. The construction method according to claim 1, characterized in that, The injection site is selected from at least one of the following: the groin area, abdomen, and back of the neck of the mouse.

7. The construction method according to claim 6, characterized in that, The injection site is the back of the neck.

8. The use of the mouse model constructed by the construction method according to any one of claims 1-7 in screening drugs for the treatment and / or prevention of intestinal damage in Sjögren's syndrome.

9. A method for screening drugs for treating and / or preventing intestinal damage in Sjögren's syndrome, characterized in that, The method includes: (1) administering the test drug to the mouse model of intestinal damage of Sjögren's syndrome constructed by the construction method of any one of claims 1-7; (2) analyzing and evaluating the therapeutic effect of the test drug, and selecting the test drug that can significantly improve the pathological characteristics of the mouse model of intestinal damage of Sjögren's syndrome.

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