Establishment method and application of peritoneal adhesion tissue model

Abstract

The invention discloses an establishment method and application of a peritoneal adhesion tissue model, and belongs to the technical field of drug screening model construction. The establishment method comprises the following steps: (1) flushing animal abdominal wall muscle tissue strips with a sterile buffer solution for later use; (2) scraping the peritoneal tissue of the abdominal wall muscle tissue strip by using a tool for 10-40 times; (3) dropwise adding absolute ethyl alcohol at the peritoneal injury tissue, and standing for 2-10 minutes; (4) dripping animal blood to the peritoneal injury tissue; (5) after the blood is coagulated, folding the abdominal wall muscle strip to fit the peritoneal surface; (6) binding up abdominal wall muscle strip tissues; (7) soaking the bound abdominal wall muscle strips in a tissue culture medium for culturing; and (8) culturing for 60-80 hours to obtain the peritoneal adhesion tissue model. The procyanidine substance capable of remarkably preventing postoperative peritoneal adhesion is screened out through the model, so that the peritoneal adhesion tissue model provides a new way for research and development of drugs for preventing postoperative peritoneal adhesion.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug screening model construction, and in particular relates to a method for establishing a peritoneal adhesion tissue model and its application. Background Art

[0002] Peritoneal adhesions are pathological connections between abdominal organs or tissues and the peritoneum. They are common complications after abdominal surgery, with an incidence rate of up to 93%. Peritoneal adhesions often involve the intestines, omentum, uterus, liver and other organs, causing chronic pain, intestinal obstruction, infertility, diarrhea and even death. Due to the high incidence and complications of peritoneal adhesions, they will still recur after adhesion lysis surgery, resulting in prolonged hospital stay and a heavy disease burden. The formation mechanism of peritoneal adhesions is complex. The main causes reported so far include trauma, bleeding, infection, foreign bodies, etc. The fibrinolytic system and immune system are widely involved in the adhesion formation process.

[0003] Common methods for preventing postoperative peritoneal adhesion formation in clinical practice include delicate surgical operations to reduce peritoneal damage, drugs, and physical barrier materials. With the progress of research, a large number of new drugs and new materials have shown excellent anti-adhesion effects in cell and animal models. At present, HMrSV5 human peritoneal mesothelial cells and primary fibroblasts extracted from the peritoneum can be used to screen effective anti-adhesion drugs, but single cell culture often ignores the role of neighboring cells and extracellular matrix, and cells are easily differentiated and changed with the increase in the number of passages, which cannot reflect the effect of drug application in vivo. Therefore, the establishment of an animal model of peritoneal adhesion is crucial for the development of new drugs. Commonly used animal models of peritoneal adhesion include abdominal wall defect-cecal injury adhesion model, abdominal wall ischemia button model, and uterine horn injury-abdominal wall defect model. The above models are all implemented in animals and are complicated to operate. Generally, stable adhesions can be formed 7 days after surgery, and a second laparotomy is often required to confirm the formation of adhesions. In addition, the components of animals are complex and vary greatly from individual to individual, so a large number of animal models are often needed to evaluate the effects of drugs and anti-adhesion materials. Furthermore, the verification of the anti-adhesion effects of different drug concentrations and different material components further increases the number of animals and experimental costs. Therefore, it is urgent to establish a new peritoneal adhesion model to overcome the above-mentioned shortcomings of cell and animal models, so as to explore the pathological mechanism of adhesion formation and evaluate the effects of drugs and materials in preventing postoperative peritoneal adhesion formation. Summary of the invention

[0004] The present invention provides a method for establishing a peritoneal adhesion tissue model, the method comprising the following steps:

[0005] (1) The animal abdominal wall muscle tissue strips are rinsed with sterile buffer and set aside;

[0006] (2) Use the tool to scrape the abdominal wall muscle tissue strips and peritoneal tissue 10-40 times;

[0007] (3) Add anhydrous ethanol to the damaged peritoneal tissue and let it stand for 2-10 minutes;

[0008] (4) dripping animal blood onto the damaged peritoneal tissue;

[0009] (5) After the blood has coagulated, fold the abdominal wall muscle strip in half so that the peritoneal surface is close together;

[0010] (6) Bandaging the abdominal wall muscle strips;

[0011] (7) immersing the bandaged abdominal wall muscle strips in tissue culture medium for culture;

[0012] (8) The peritoneal adhesion tissue model can be obtained after 60-80 hours of culture.

[0013] Preferably, the sterile buffer in step (1) is a sterile phosphate buffer solution containing 1% penicillin and 1% streptomycin.

[0014] More preferably, in step (2), the abdominal wall muscle tissue strips and peritoneal tissue are scraped 20-30 times.

[0015] More preferably, the step (3) is performed by standing for 5 minutes.

[0016] More preferably, the pressure when bandaging the abdominal wall muscle strip tissue in step (6) is 10 to 15 mmHg.

[0017] More preferably, the tissue culture medium in step (7) is DMEM culture medium.

[0018] More preferably, the DMEM culture medium contains 0.01% insulin, 1% penicillin, and 1% streptomycin.

[0019] More preferably, the culture time in step (8) is 72 hours.

[0020] The present invention also provides the use of the peritoneal adhesion tissue model constructed by the above-mentioned establishment method in screening drugs for preventing the formation of postoperative peritoneal adhesions.

[0021] The present invention also provides the use of proanthocyanidins in preparing a product for preventing postoperative peritoneal adhesion formation.

[0022] Compared with the disclosed invention patent ZL201910286351.6, the present invention uses animal abdominal wall tissue ex vivo culture to construct an in vitro peritoneal adhesion model by quantifying the degree of abdominal wall abrasion, coagulation volume and pressurization pressure. The model can be constructed using multiple peritoneal tissue strips from the same mouse, which reduces the experimental cost. Adhesion bands can be formed after 72 hours of culture; and the anti-adhesion effects of multiple drug doses or different material components can be tested in the same individual tissue; the tissue model is also convenient for daily monitoring of adhesion formation and collection of specimens. At the same time, the present invention applies the above-mentioned peritoneal adhesion tissue model to the screening of new drugs for preventing postoperative peritoneal adhesions. The research results show that proanthocyanidins have a significant effect in preventing adhesion formation. The establishment of this peritoneal tissue adhesion model provides a new way to develop drugs for preventing postoperative peritoneal adhesions.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] 1) The present invention provides a method for establishing an ex vivo peritoneal adhesion tissue model. During the modeling process, the size of the peritoneal tissue strip, the volume of the blood clot, and the degree of tissue bandage pressure are quantified for the first time, thereby improving the success rate of the in vitro model and reducing the differences in adhesions caused by subjective modeling;

[0025] 2) The present invention provides a method for establishing an ex vivo peritoneal adhesion tissue model. During the modeling process, multiple peritoneal tissue strips from the same animal can be used for repeated construction and experiments, thereby reducing individual differences and reducing experimental costs;

[0026] 3) The present invention provides a method for establishing an ex vivo peritoneal adhesion tissue model, which can form adhesions after 72 hours of in vitro culture, saving the time for adhesion formation (general animal models take up to 7 days), and facilitating daily assessment of adhesion formation;

[0027] 4) The method for establishing an in vitro peritoneal adhesion tissue model provided by the present invention can be used to screen new drugs for preventing postoperative peritoneal adhesion;

[0028] 5) The proanthocyanidins described in the present invention have a good effect in preventing peritoneal adhesions, and provide a new drug option for preventing the formation of postoperative peritoneal adhesions. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a real picture of the modeling in Example 1.

[0030] Figure 2 This is a real picture of proanthocyanidins sprayed on the peritoneal wound in Example 2.

[0031] Figure 3 This is a photo of the actual results of the application of proanthocyanidins in Example 2 to prevent peritoneal adhesions.

[0032] Figure 4 This is the result of H&E staining of proanthocyanidins applied to prevent peritoneal adhesion in Example 2.

[0033] Figure 5 These are the results of detecting the mRNA expression levels of TNF-α, t-PA and PAI-1 after proanthocyanidins were applied to peritoneal adhesion to prevent adhesion tissue in Example 2. DETAILED DESCRIPTION

[0034] Example 1 Establishment of peritoneal adhesion tissue model

[0035] 1. Experimental process

[0036] 1.SPF grade, SD strain rats weighing 220-250g.

[0037] 2. Adapt the animals to the appropriate diet and water in a standardized animal room for 3 days with 12 hours of light and 12 hours of dark each day.

[0038] 3. Before modeling, the rats were weighed and injected intraperitoneally with 2 mL / kg of 3% sodium pentobarbital solution. The SD rats were anesthetized and fixed in a sterile animal operating table. The abdomen was depilated and skinned to a size of 4×5 cm. The skin was disinfected with 75% alcohol and 1% iodine in sequence and covered with a sterile sheet.

[0039] 4. Use sterile ophthalmic scissors to make a 4×5 cm abdominal U-shaped incision layer by layer along the midaxillary line on the right side of the rat.

[0040] 5. Use tissue forceps to pull and completely expose the abdominal wall tissue. Carefully peel off the skin tissue and keep the remaining full-thickness abdominal muscle tissue for later use.

[0041] 6. Cut 4 to 5 abdominal wall muscle tissue strips, each with a size of 1×3 cm, and rinse them three times with a sterile phosphate buffer solution containing 1% penicillin and 1% streptomycin at 4°C for later use.

[0042] 7. Flatten the abdominal wall muscle tissue strip, and use a No. 10 sterile scalpel to scrape the peritoneal tissue 20 to 30 times along the long axis of the abdominal wall muscle tissue strip. The scraped peritoneal tissue area is 1×2 cm, and the scraped peritoneal tissue thickness is 0.1 to 0.2 cm.

[0043] 8. Pipette 50-100 μL of anhydrous ethanol and drop it on the damaged peritoneal tissue. Let it stand for 5 minutes to induce chemical injury.

[0044] 9. Draw another 200 μL of rat blood and drop it on the injured peritoneal tissue. After standing for 10 to 15 minutes, observe the blood coagulation to form a blood clot.

[0045] 10. Use sterile ophthalmic forceps to fold the abdominal wall muscle tissue strip in half to fit the peritoneal wound surface.

[0046] 11. Use sterile gauze with a size of 7.5×7.5cm to apply pressure to the abdominal wall muscle strips in 2 to 3 layers with a bandage pressure of 10 to 15 mmHg.

[0047] 12. Place the bandaged abdominal wall muscle strip tissue in a sterile tissue box with dimensions of 4×2.6×1.2 cm.

[0048] 13. Add 15-20 mL of DMEM culture medium (added with 0.01% insulin, 1% penicillin, and 1% streptomycin) to a sterile cell culture dish with a diameter of 10 cm, immerse the tissue cassette in the dish, and observe the peritoneal adhesion after 72 hours of culture.

[0049] 14. Finally, 3 mL of 3% sodium pentobarbital solution was injected into the abdominal aorta to euthanize the animal.

[0050] 2. Experimental Analysis

[0051] Figure 1 Figure 2: Actual modeling: (a) The number of abdominal wall muscle tissue strips separated and cut; (b) Actual picture of peritoneal scraping injury; (c) 200 μL of rat blood was aspirated and dripped on the injured peritoneal tissue; (d) Blood formed a blood clot at the peritoneum; (e) The abdominal wall muscle tissue strip was folded in half to make the peritoneal wound fit; (f) The abdominal wall muscle strip wrapped with sterile gauze was placed in a tissue box; (g) The tissue box was immersed in a culture dish for culture; (h) Peritoneal adhesion was observed after 72 hours of culture (indicated by the arrow).

[0052] Example 2 Application of peritoneal adhesion tissue model in screening new drugs for preventing postoperative peritoneal adhesion formation

[0053] 1. Experimental process

[0054] The method for establishing the peritoneal adhesion tissue model in this embodiment is the same as that in Example 1, except that step 10 is performed according to the following process: the abdominal wall muscle tissue strips are randomly divided into four groups, A and B, with group A being the positive control group, group B being the group receiving a low concentration of proanthocyanidins (C 30 H 26 O 12 ) B2 group, group C was a medium concentration proanthocyanidin B2 group of 2.5 mg / kg (tissue weight), group D was a high concentration proanthocyanidin B2 group of 5 mg / kg (tissue weight), and groups BD were respectively treated with proanthocyanidin powder applied to the peritoneal damaged tissue (such as Figure 2 shown).

[0055] 2. Experimental Analysis

[0056] 1. As Figure 3As shown, 72 hours after modeling, compared with the positive control group, groups C and D did not form obvious peritoneal adhesions, while the low concentration of 1 mg / kg proanthocyanidin B2 in group B did not effectively prevent the formation of peritoneal adhesions, and dense adhesion bands were formed. The above results suggest that the medium concentration of 2.5 mg / kg proanthocyanidin B2 treatment can effectively prevent the formation of peritoneal adhesions.

[0057] 2. If Figure 4 As shown, 72 hours after modeling, the peritoneal adhesion tissue was taken for H&E staining. The results showed that compared with the positive control group and the low-concentration proanthocyanidin B2 group, the 2.5 mg / kg medium-concentration proanthocyanidin B2 group and the 5 mg / kg high-concentration proanthocyanidin B2 group did not form obvious pathological adhesions, the peritoneum was relatively smooth and intact, and no inflammatory cell infiltration was observed in the peritoneal tissue.

[0058] 3. Such as Figure 5 As shown, 72 hours after modeling, the nucleic acid of peritoneal adhesion tissue was extracted, and the mRNA levels of TNF-α, t-PA, and PAI-1 were detected. The results showed that the medium concentration of 2.5 mg / kg proanthocyanidin B2 group and the high concentration of 5 mg / kg proanthocyanidin B2 group could prevent peritoneal adhesion formation by reducing the expression levels of TNF-α and PAI-1 and up-regulating the expression of t-PA.

[0059] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for establishing a peritoneal adhesion tissue model, characterized in that: The establishment method comprises the following steps: (1) The animal abdominal wall muscle tissue strips are rinsed with sterile buffer and set aside; (2) Use the tool to scrape the abdominal wall muscle tissue strips and peritoneal tissue 10-40 times; (3) Add anhydrous ethanol to the damaged peritoneal tissue and let it stand for 2-10 minutes; (4) dripping animal blood onto the damaged peritoneal tissue; (5) After the blood has coagulated, fold the abdominal wall muscle strip in half so that the peritoneal surface is close together; (6) Bandaging the abdominal wall muscle strips; (7) immersing the bandaged abdominal wall muscle strips in tissue culture medium for culture; (8) The peritoneal adhesion tissue model can be obtained after 60-80 hours of culture.

2. The establishment method according to claim 1, characterized in that: The sterile buffer in step (1) is a sterile phosphate buffer solution containing 1% penicillin and 1% streptomycin.

3. The establishment method according to claim 2, characterized in that: In the step (2), the abdominal wall muscle tissue strips and the peritoneal tissue are scraped 20-30 times.

4. The establishment method according to claim 3, characterized in that: The step (3) is allowed to stand for 5 minutes.

5. The establishment method according to claim 4, characterized in that: The pressure when bandaging the abdominal wall muscle strip tissue in step (6) is 10 to 15 mmHg.

6. The establishment method according to claim 5, characterized in that: The tissue culture medium in step (7) is DMEM culture medium.

7. The establishment method according to claim 6, characterized in that: The DMEM culture medium contains 0.01% insulin, 1% penicillin and 1% streptomycin.

8. The establishment method according to claim 7, characterized in that: The culture time in step (8) is 72 hours.

9. Use of the peritoneal adhesion tissue model constructed by the establishment method according to any one of claims 1 to 8 in screening drugs for preventing postoperative peritoneal adhesion formation.

10. The application of proanthocyanidins in the preparation of products for preventing postoperative peritoneal adhesion formation.

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