动物造模,药效评价,萎缩性胃炎 z-bio 2024-08-21 616

　　(1) Copy method

　　1) Mouse model: BALB/c mice, SPF grade, 6-8 weeks old. After fasting for 12 hours and water deprivation for 8 hours, inoculate Hp (SS1) culture suspension at 1000000000000 CFU/L once every other day, for a total of 3 times, with 100 μ l per time. Preparation method of Hp (SS1) culture suspension: Hp (SS1) strain was inoculated on Burcella broth agar medium containing 10% sheep blood and multiple antibiotics (10 μ g/ml vancomycin, 3 μ g/ml polymyxin, 5 μ g/ml amphotericin B, 5 μ g/ml sulfonamide enhancer), cultured at 37 ℃, 5% O2, 10% CO2, 85% N2 for 48 hours, then washed with PBS and selected for dynamic detection. At 12 and 24 weeks after inoculation with Hp, the patient was euthanized by cervical dissection, and the stomach was cut open along the greater curvature of the stomach. The stomach contents were washed away with PBS, and the tissue from the esophagus to the duodenal bulb was cut along the lesser curvature of the stomach. The tissue was fixed with 10% formaldehyde, embedded in paraffin, sliced at 5 μ m, stained with HE to observe inflammation, and stained with Giemsa to observe Hp colonization.

　　2) Rat model: Young Wistar rats weighing 110-130g were fasted for 12 hours and orally administered with 2ml of 5% Na2HCO3 solution per rat. After 15 minutes, 2ml of a mixed strain of 1000000000000 CFU/L Hp (S67 and S73) was administered per rat. Infection once a week for 4 times. One week after the last infection, the animals were euthanized and their stomachs were opened. The stomachs were cut along the greater curvature of the stomach and washed with PBS to remove gastric contents. Five tissues were taken from the relatively fixed part of the pylorus for direct smear, urease test, and Hp culture. The remaining stomach bodies were fixed with 10% formaldehyde for histopathological examination.

　　3) Mongolian sand rat model: Mongolian sand rats (MG), 8 weeks old, were fasted and deprived of water for 12 hours before being orally administered with 50% ethanol at a dose of 0.3ml/animal. After that, they continued to be fasted and deprived of water. The next day, they were orally administered with 1000000000000 CFU/L Hp bacterial solution at a dose of 0.5ml/animal, once in the morning and once again in the morning. The model was completed at 12 weeks. Before euthanizing the model animals, they were fasted and dehydrated for 24 hours. Prior to euthanization, blood was drawn from the eyeballs, serum was separated, and anti Hp antibodies were detected by ELISA. After euthanization, the stomach was opened and cut along the greater curvature of the stomach. The residual gastric tissue was gently washed away with physiological saline, and the overall condition of the gastric mucosa was observed with the naked eye. Then, half of the mucosal tissue was cultured, smeared, and subjected to rapid urease paper test. The other half of the mucosal tissue was fixed with 10% formaldehyde for histopathological examination.

　　4) Small pig model: After the onset of labor symptoms in the full-term pregnant Chinese No.1 small sow, the fetus was removed by caesarean section in the operating room, and the suckling pig was placed in a sterile isolation room and given sterile milk for free feeding. At 10 days, orally administer 12.5mg/animal of Indomethacin once a day for 3 consecutive days. On the 4th day, orally administer 2ml of 1000000000000 CFU/L Hp bacterial solution once a day for a total of 3 times. Then, the animals were transferred to the secondary environment of experimental animals and euthanized 30 days after Hp administration. Esophageal, gastric body, gastric antrum, and duodenal mucosal specimens were collected for tissue sectioning and HE staining to observe histological changes. Urease method was used to detect Hp bacteria, Warthin Starry silver staining was used to detect Hp bacteria, and microaerophilic method was used to culture Hp bacteria.

　　(2) Model characteristics: At 12 weeks of infection in model mice, a large amount of Hp colonization was observed in the mucus on the surface of gastric tissue and the gastric tissue at the top of the gastric pit, especially at the junction of the gastric body antrum and the gastric body fundus. At 24 weeks, Hp colonization density increased; In addition to atrophy (glandular distortion and structural abnormalities) and degeneration of gastric epithelial cells, gastric tissue is characterized by infiltration of inflammatory cells; At 12 weeks of infection, the infiltration of inflammatory cells was mainly composed of multinucleated lymphocytes, and at 24 weeks, there was also infiltration of neutrophils and eosinophils. At 4 weeks of MG infection, Hp infection can be detected as positive, and at 12 and 24 weeks, the infection rate reaches 100%; Macroscopic examination shows obvious bleeding, chronic active gastritis, and ulcers in the gastric mucosa, and sometimes ulcers can penetrate deep into the muscle layer; Microscopic examination showed that at 4 weeks of infection, a large number of inflammatory cells, including neutrophils, lymphocytes, and monocytes, appeared in the epithelial cells of the gastric antrum and pylorus, as well as between the epithelial cells of the glandular fossa and in the lamina propria. The submucosal blood vessels were dilated and congested, and the glands were atrophied with epithelial cell degeneration and necrosis. With the passage of time, the infiltration of inflammatory cells intensifies, gradually forming lymphoid follicles dominated by lymphocyte aggregation. A large amount of Hp can be clearly observed on the surface mucus of the gastric antrum, pylorus, and gastric body mucosal epithelial cells, in the gastric glandular fossa, and between epithelial cells. At 30 days of infection in model miniature pigs, the surface of the gastric mucosa showed slight thickening and edema, and there were varying degrees of superficial mucosal erosion and inflammatory exudation in the esophageal, gastric, and duodenal mucosa. There was infiltration of inflammatory cells in the lamina propria, and some lymphoid follicular hyperplasia was observed, presenting pathological features of chronic active gastritis. Bacteriological examination showed that Hp infection was all positive in the mucosa of the esophagus, stomach body, antrum, duodenum, small intestine, and large intestine. Warthin Starry silver staining revealed a large number of rod-shaped or S-shaped Hp bacteria stained black on the surface of the stomach body and antrum mucosa. Accumulated Hp could be seen in some lesions, and clear Hp stained black brown could also be seen in the esophageal mucosa and duodenal mucosa.

　　(3) A large amount of epidemiological data in comparative medicine confirms that Hp is the main cause of non erosive gastritis, including chronic atrophic gastritis. Hp is a spiral shaped, Gram negative microorganism that can cause the vast majority of non erosive gastritis and its complications. Human infection with this bacterium can cause inflammation of the gastric mucosa, thereby affecting the secretion mechanism of the stomach. Make the gastric mucosa more sensitive to acid damage. In recent years, researchers have been trying various methods to infect different animals with Hp, aiming to establish a human like animal model of chronic atrophic gastritis induced by Hp infection, and have established the Luoshuang standard. This standard requires: ① Hp colonization can be seen on the gastric mucosa, and pathological changes similar to those in humans can be observed in the gastric body and antrum. ② There is a certain amount of Hp per gram of gastric tissue To observe the adhesion between Hp and gastric mucosa Long term and persistent bacterial infections are necessary The strain used must be a stable strain that can still infect animals after a certain number of passages in vitro. According to this standard, researchers have successively infected and successfully induced chronic gastritis animal models in mice, rats, Mongolian sand rats, dogs, and pigs with Hp. However, for most animals, the low colonization rate and short duration of infection remain insurmountable technical barriers that affect the success rate of Hp modeling. Therefore, selecting susceptible animals to Hp and screening for Hp strains with strong motivation are the technical keys. For example, Hp (SS1) is a strain of bacteria suitable for colonization in the stomach of mice that has been continuously passaged and isolated. This strain has extremely strong dynamics and contains CagA and VagA, belonging to type I Hp. Epidemiological investigations have shown a close relationship between type I Hp and the occurrence of peptic ulcers, which contain pathogenic islands (Pathogenicity Island), express vacA, and have positive vacuolar toxicity. Inoculating this strain into the stomach of SPF grade BABA/c mice can cause severe pathological changes in animal stomach tissue, including disappearance of normal gastric glands, erosion, and ulcers, with a success rate of 100%. Compared with mice, rats are less likely to develop Hp infection in the absence of gastric mucosal damage. Generally, it is necessary to first cause gastric mucosal damage in rats, and then infect them with Hp. Therefore, it is rare to use Hp alone to prepare a rat model of chronic gastritis. MG itself has a low infection rate with Hp, but pre subcutaneous injection of 20mg/kg body weight of indomethacin or oral administration of 5ml/kg body weight of 50% ethanol can increase the infection rate to over 70% and cause a significant gastritis model.However, for most animals, the low colonization rate and short duration of infection remain insurmountable technical barriers that affect the success rate of Hp modeling. Therefore, selecting susceptible animals to Hp and screening for Hp strains with strong motivation are the technical keys. For example, Hp (SS1) is a strain of bacteria suitable for colonization in the stomach of mice that has been continuously passaged and isolated. This strain has extremely strong dynamics and contains CagA and VagA, belonging to type I Hp. Epidemiological investigations have shown a close relationship between type I Hp and the occurrence of peptic ulcers, which contain pathogenic islands (Pathogenicity Island), express vacA, and have positive vacuolar toxicity. Inoculating this strain into the stomach of SPF grade BABA/c mice can cause severe pathological changes in animal stomach tissue, including disappearance of normal gastric glands, erosion, and ulcers, with a success rate of 100%. Compared with mice, rats are less likely to develop Hp infection in the absence of gastric mucosal damage. Generally, it is necessary to first cause gastric mucosal damage in rats, and then infect them with Hp. Therefore, it is rare to use Hp alone to prepare a rat model of chronic gastritis. MG itself has a low infection rate with Hp, but pre subcutaneous injection of 20mg/kg body weight of indomethacin or oral administration of 5ml/kg body weight of 50% ethanol can increase the infection rate to over 70% and cause a significant gastritis model. The histological manifestations include mucosal erosion within 3 weeks, infiltration of neutrophils and monocytes in the mucosal layer, lymphoid follicles in the submucosal and submucosal layers at 6 weeks, and atrophy, intestinal metaplasia, and ulceration at 12-24 weeks. It is worth mentioning that Hp infection with MG can persist in the stomach for a long time, mainly existing in the mucous layer on the surface of the stomach, with a few adhering to the surface of mucous cells. The amount of bacteria in MG stomach can reach 100000 CFU, and the lesions caused are mainly in the gastric antrum, similar to humans. According to the Luoshuang standard, except for ③ which is not very typical, all others meet this standard. In addition to the successful infection of Hp in the aforementioned animals, successful replication of Hp infection models using animals such as Xisheng dogs, Xisheng pigs, and pigs without specific pathogens has also been achieved abroad. Due to the omnivorous nature of pigs, their stomach anatomy, physiological functions, and dietary habits are very similar to those of humans. Therefore, choosing pigs as experimental animals has its reasonable side. However, due to the complexity of the model replication technology, high environmental requirements, and expensive modeling costs, it is rare to see practical applications. In this context, some researchers attempted to establish Hp infection under the conditions of Xisheng pigs first, and then raised the animals under normal conditions. As a result, Hp could still persist, thus effectively solving the bottleneck problems of technology and price that are inevitably encountered in the full process experiment of Xisheng pigs.Experiments have shown that after infection with Hp, model animals first cause transient and discontinuous neutrophil infiltration in non glandular areas of the cardia, followed by lymphocyte infiltration in glandular areas of the stomach; Lymphocytes initially aggregate only in the submucosal and mucosal layers, and then develop into disjointed lymphoid follicles in the submucosal and lamina propria layers. Warthin Starry silver staining of gastric mucosal tissue revealed that Hp is located between the gastric mucosal epithelium and the protective layer of the surface mucus, which is similar to the expression of Hp in the human stomach. The key technology for replicating the Hp model of China's No.1 miniature pig mainly lies in orally administering nonsteroidal drug indomethacin for pre-treatment before Hp inoculation. Due to the presence of a large amount of prostaglandins (PG) in the gastrointestinal tract, especially in the gastric mucosal layer, the latter has a wide range of regulatory effects on the physiological functions of the gastrointestinal tract, including affecting the secretion of gastric mucus, regulating the blood flow of the gastric mucosa, and protecting the barrier function of the mucosa. Indomethacin can inhibit prostaglandin (PG) synthesis in the body. When indomethacin is ingested into the body, it leads to a significant decrease in PG content, especially in the gastric mucosa, which directly damages the barrier function of the gastric mucosa and reduces the protective function of epithelial cells, thus creating suitable conditions for Hp to settle in the gastric mucosa.