动物实验,胃黏膜缺损 z-bo 2020-09-04 481

　　Background: The gastric mucosal barrier is a complex system involving physical, chemical and biological defense mechanisms to protect the stomach from irritating food, hydrochloric acid and pepsin activity. Several conditions, such as gastritis, gastric mucosal erosion and ulcers, can disrupt the gastric mucosal barrier and cause its damage. Gastric mucosal injury is a common disease in veterinary medicine. Because of many frequently used drugs, such as non-steroidal anti-inflammatory drugs or glucocorticoids. There are several diseases that can destroy mucosal defense mechanisms including Helicobacter pylori infection, liver or kidney disease, adrenal insufficiency, shock, spinal cord disease, autoimmune disease, primary gastrointestinal disease, and tumors. Therefore, gastric or duodenal ulcers are usually caused by defects in the gastric mucosa or duodenal epithelial barrier function. The gastric ulcer is described as a huge "moon pit" defect in the gastric mucosa. Endoscopic studies showed that 48.5% of dogs had ulcers on the proximal end of the stomach or duodenum. Compared with other domestic animals, gastric cancer is also very common in gastric cancer, because tumor resection will cause deep wounds in the stomach, requiring tissue reconstruction. Most gastric malignancies are cancer, accounting for 50-90%. Followed by sarcoma and malignant lymphoma. Some studies have tried to transplant gastric mucosal defects. Amniotic membrane is a promising new biological material. AM is the innermost layer of the fetal membrane. In addition to collagen sponge IV, V, and VII, there is also a layer of epithelial cells. In addition to fibronectin and laminin containing mesenchymal cells, there is a thicker basement membrane. AM is considered to be an ideal tissue for allogeneic transplantation due to its low immunogenicity. In the process of allogeneic transplantation, AM has the ability to inhibit T lymphocytes. As a basement membrane, AM promotes the migration of epithelial cells, enhances the adhesion of basal epithelial cells, promotes epithelial cell differentiation, and prevents epithelial cell apoptosis. Amniotic membrane cells release cytokines related to wound healing, including platelet-derived growth factor, vascular endothelial growth factor, angiogenesis factor, transforming growth factor β2 (TGF-ββ2), tissue inhibitor of matrix metalloproteinase 1 (TIMP-1 and TIMP- 2). AM also has anti-inflammatory, anti-fibrotic, anti-angiogenic and antibacterial properties. It induces down-regulation of TGF-β signaling and is responsible for the activation of fibroblasts during wound healing. The application of AM on wounds also significantly relieves painful burns due to the adhesion of wounds and skin nerve endings coverage. Many studies have shown that cells from AM can differentiate into various mature cells, including adipocytes, bone cells, chondrocytes, muscle cells, cardiomyocytes, liver cells, nerve cells, and vascular endothelial cells. These observations indicate that AM contains stem cell-like cells that can provide an alternative cell source for regenerative medicine. AM has been used in reconstructive surgery for nearly a century. It is used for burns and ulcers, such as wound biological dressings, such as ophthalmic surgical transplants. So far, human transplant tissues are stored in glycerol solution or cryopreserved. Recent studies have shown that amniotic membrane epithelial cells cannot survive after preservation. Currently It is not clear whether growth factors can be stored by freezing. The purpose of this study is to describe a simple, novel, inexpensive and effective surgical method to repair gastric mucosal defects in dogs with fresh allografts.

　　Method: All operations are performed under general anesthesia, and all efforts are made to reduce animal suffering and reduce the number of animals used. Six adult male breed dogs, approximately 3–5 years old, weighing 20–25 kg. The animals were divided into two groups (three in each group). In the AM group, dogs underwent surgery for gastric mucosal defects and then treated with fresh allografts. In the positive (C + VE) control group and dogs, the same operation was performed and traditional medication was used.

　　Preparation of amniotic membrane: Take fresh placental specimens through cesarean section, and keep intact fetal membranes. Then rinse with sterile saline solution to remove the peeled debris. The placenta was rinsed thoroughly with sterile normal saline containing 100 U/ml penicillin, 0.2 mg/ml streptomycin and 0.025 mg/ml amphotericin B. Then the amniotic membrane was washed several times with normal saline, and the AM was placed in a petri dish containing the same mixture of saline, and then stored in a refrigerator at 4 °C for use within a week.

　　Induction of gastric mucosal defect and amniotic membrane transplantation: Each dog was given atropine sulfate and xylazine, and then ketamine hydrochloride was used to induce and maintain anesthesia. The selected dogs were subjected to laparoscopic surgery on the midline of the abdomen, and a 3 cm × 2 cm round patch was applied to the gastric mucosa surface. In the AM group, the induced ulcer was repaired by 4 cm ×5 cm fresh double layer  AM. The first layer of suture gastric mucosal wound was sutured with Vicryl 3/0 and the epithelial side simply and continuously, and then the second layer was sutured in the same way. In the C + VE group, a round patch has no coverage. The open surgery wound was sutured in 2 layers. Animals are treated with traditional medicine. Animals receive proton pump inhibitor drugs and mucosal coaters.

　　Postoperative care: Avoid the stimulation of gastric acid and pepsin secretion, and fast for the first 3 days after surgery. Then feed the animals with cooked rice and chicken. Both AM group and C + VE group were given metronidazole and flumox drops for 7 days to avoid Helicobacter infection. Sutures were removed 10 days after surgery.

　　Clinical evaluation: This study conducted daily health checks on animals, such as body temperature, heart and respiratory rate, mucous membrane color, lymph node size, desire to eat, urination and defecation, and personality.

　　Endoscopic evaluation: All dogs of the two groups of animals were anesthetized with anesthetic at 10 and 21 days after surgery. Use an endoscope with video for inspection.

　　Sample: Blood sample: blood is collected from the jugular vein; one part is stored in a tube containing EDTA, agglutinated with the other part and centrifuged at 3500 rpm for 15 minutes.

　　Gastric juice: Samples are collected through a catheter for endoscopic evaluation. At 10 and 21 days after the operation, samples of the two groups were collected and stored at -80 °C for analysis of biochemical and pepsin concentrations.

　　Surgical biopsy: 21 days after the operation, two groups of animals were collected by gastrostomy for histopathological and immunohistochemical evaluation.

　　Hematological analysis: White blood cell image: Identification of white blood cell counts are analyzed by traditional methods as follows: a drop of blood is scattered on a glass slide, the blood membrane is fixed with methanol for 2 minutes, stained with Giemsa diluted 1:9 for 8-10 minutes, then washed and dried. , Under the oil lens, by moving the lens in the area including the center and the periphery, the area where the cell shape is clearly visible is selected. A total of 200 cells are counted, and each white cell is recorded.

　　Biochemical analysis: Estimate total protein and albumin levels according to the method described by doumas. Serum globulin was calculated according to Lanter. Use the kit to determine the triglyceride concentration.

　　Evaluation of gastric juice pepsin activity: Using casein as the substrate, the gastric juice pepsin activity was determined by the method described by Hawke et al. Extract 1 ml from different concentrations of bovine pepsin, from 0.1 to 1 mg/100 ml 0.1 N HCl , Was transferred to a test tube and incubated in a 3.9 ml water bath at 37 °C for 30 min. Add 10 ml TCA to the tube and keep it upright for 10 minutes and then filter. The blank control is to add 10ml of TCA to each concentration before adding enzyme. The optical density of the filtrate was measured at a wavelength of 280 nm. For the determination of the proteolytic enzyme activity secreted by the stomach, the same procedure was performed at a concentration of 2% 0.1 N HCl.

　　Histopathological evaluation: At the end of the experiment, samples were collected from gastric mucosal defects and grafts in the two groups, and fixed in formalin for 48 hours. These biopsies are then processed using paraffin embedding technology. After slices with a microtome to a thickness of 5 μm, the HE stained microscopy. The two groups were evaluated for ulcers caused by re-epithelialization and leukocyte infiltration. 0～3 lymphocytes were used for semi-quantitative analysis of inflammation. The inflammation in the ulcer area is scored by lymphocyte count in three areas (200× magnification) (semi-quantitative score: 0, < 5% 25="" cells="">50%). Masson's trichrome staining was performed on the paraffin-embedded tissue block sections to detect fibrosis.

　　Results: Clinical findings: The study animals did not show health indicators such as body temperature, heart rate and respiration, mucous membrane color, lymph node size, feeding appetite, and urination and defecation frequency and personality disorders.

　　Results of endoscopic and surgical exploration: On the tenth day after operation in the C + VE group, gastric endoscopy revealed that a blood clot was covering the mucosal defect. In the AM group, the amniotic membrane covered the mucosal defect, there was no inflammation, and there was no bleeding around the mucous membrane (mucosal part). In the C + VE group, gastroendoscopy on the 21st day after surgery showed small round ulcers, mild bleeding, and surrounding mucosal inflammation. The same mucosal defect was not found in the AM group, and no AM was detected on the mucosal surface. There is also no sign of inflammation or bleeding.

　　Hematology findings: A complete blood picture showed a slight decrease in red blood cells in the control group at 10 and 21 days after surgery. A severe drop in HGB in the control group indicates anemia, but it is in the normal range in the AM group. There was no difference in white blood cells between the two groups.

　　Biochemical examination results: On the tenth day after surgery, the serum total protein concentration (g%) and globulin concentration (g%) of the C + VE group were significantly increased compared with the preoperative and AM groups. Serum TAG concentration and serum cholesterol concentration increased significantly on the 10th day after surgery, and then gradually decreased. On the 21st day, the serum concentration was the same as the AM group.

　　Pepsin activity: The pepsin activity of the C+VE group increased significantly at 10 days after surgery, and the activity of the AM group decreased significantly at 10 and 21 days.

　　Histopathological and immunohistochemical results: The histopathological examination of the AM group revealed normal tissue structure. With MTC staining, loose connective tissue was stained blue and fibroblasts stained red. Collagen cells are not stained. In the C + VE group, the epithelial cells of the gastric mucosa did not regenerate and left hemorrhagic ulcers. In addition, mononuclear inflammatory cells were observed in the surrounding connective tissue. Gastric ulcers in the AM group showed almost complete gastric epithelial regeneration, except for tiny areas not covered by epithelium.

　　Conclusion: It can be concluded from this study that the use of fresh allogeneic materials to repair canine gastric mucosal defects has shown great influence in order to optimize the structure of gastric mucosa and normal gastric mucosa before, after, and after epithelium. The repair of the barrier provides a new method.