动物模型,慢性萎缩性胃炎 z-bo 2022-02-05 435

　　(1) Copy method

　　1) Mouse model: BALB/c mouse, SPF grade, 6-8 weeks old. After fasting for 12h and water-free for 8h, inoculate Hp(SS1) culture suspension 1000000000000CFU/L, once every other day, a total of 3 times, 100μl each time. The preparation method of Hp(SS1) culture suspension: Hp(SS1) strain is inoculated with 10% sheep blood and multiple antibiotics (10μg/ml vancomycin, 3μg/ml polymyxin, 5μg/ml amphotericin B , 5μg/ml sulfa synergist) on Burcella broth agar medium, culture at 37℃, 5% O2, 10% CO2, 85% N2 for 48h, then wash it with PBS, and select those who pass the power test . At 12 and 24 weeks of Hp inoculation, the neck was severed, the stomach was taken by laparotomy, cut 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 , 10% formaldehyde fixation, paraffin embedded, 5μm section, HE staining to observe the inflammatory reaction, Giemsa staining to observe the Hp colonization.

　　2) Rat model: Wistar juvenile rats, weighing 110-130g, fasted for 12 hours with 2ml of 5% Na2HCO3 solution per rat by gavage, 15min later with 1000000000000 CFU/L Hp (S67 and s73) mixed strain 2ml per rat. Infection 1 time per week for 4 times. One week after the last infection, the animals were sacrificed acutely, the stomach was taken out of the stomach, and the stomach was cut along the greater curvature of the stomach. The contents of the stomach were washed away with PBS. Five tissues were taken from the relatively fixed part of the pylorus for direct smear, urease test and Hp culture, the rest of the gastric body was fixed with 10% formaldehyde for histopathological examination.

　　3) Mongolian gerbil model: Mongolian gerbil (Mongoliangerbil, MG), 8 weeks old, fasted for 12 hours and then given 50% ethanol 0.3ml/head by gavage, then continue fasting and water-free, give 1000000000000 CFU by gavage the next day /L Hp bacterial liquid 0.5ml/piece, 1 time in the morning and afternoon, and then gavage once in the morning the next day, and the model was completed at 12 weeks. The model animals were fasted and water-free for 24 hours before being sacrificed. Before being sacrificed, the eyeballs were bled, the serum was separated, and the anti-Hp antibody was detected by ELISA. After sacrifice, the stomach was taken out and cut along the greater curvature of the stomach. The stomach residues were gently washed away with normal saline. Observe the general condition of the gastric mucosa, and then half of the mucosal tissues are cultured, smeared and fast urease paper test, and the other half of the mucosal tissues are fixed with 10% formaldehyde for histopathological examination.

　　4) Miniature pig model: After full-term pregnancy, China No. 1 miniature sows were given birth by caesarean section in the operating room. The suckling pigs were raised in a sterile isolator, and they were given sterile milk and fed freely. On the 10th day, 12.5mg of indomethacin was given orally, once a day, for 3 consecutive days, and on the 4th day, 1000000000000CFU/L Hp bacterial liquid 2ml/head was given orally, once/3d, for a total of 3 times. Then, the animals were transferred to the secondary environment of experimental animals, and they were sacrificed 30 days after the Hp administration. The esophagus, gastric corpus, gastric antrum, and duodenal mucosa specimens were taken for tissue section HE staining, and the tissues were observed Changes in science, parallel urease method to detect Hp bacteria, Warthin-Starry silver staining to find Hp bacteria, and microaerobic method to cultivate Hp bacteria.

　　(2) Model characteristics When the model mice are infected for 12 weeks, a large number of Hp colonizations can be seen in the mucus on the gastric tissue surface and the gastric tissue on the top of the gastric pit. The colonization density is the highest in the gastric body-antrum and the gastric body-fundus junction. At 24 weeks, the density of Hp colonization increased; in addition to atrophy (glandular distortion and structural abnormality) and degeneration of gastric epithelial cells, the typical features of gastric tissue were infiltration of inflammatory cells; at 12 weeks of infection, inflammatory cells infiltrated with polynuclear lymph Mainly cells, neutrophils and eosinophils infiltrated at 24 weeks. When the model MG is infected for 4 weeks, Hp infection can be detected positive, and the infection rate reaches 100% at 12 and 24 weeks; the macro examination shows obvious bleeding, chronic active gastritis and ulcers in the gastric mucosa, and sometimes deep ulcers Reach 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 mucosa of the gastric antrum and pylorus, the crypt epithelial cells and the lamina propria. Cells, submucosal blood vessels were dilated and hyperemic, and glands atrophied accompanied by epithelial cell degeneration and necrosis. As time goes by, the infiltration of inflammatory cells intensifies, and lymphoid follicles dominated by lymphocyte aggregation are gradually formed. In the gastric antrum, pylorus and gastric body mucosa epithelial cell surface mucus, gastric crypts and epithelial cells can clearly see the presence of a large number of Hp. When the model miniature pigs were infected for 30 days, the gastric mucosa surface was slightly thickened and edema, the esophagus, stomach, and duodenum mucosa had varying degrees of superficial mucosal erosion and inflammatory exudation, and inflammatory cell infiltration in the lamina propria. Part of the lymphoid follicle hyperplasia can be seen, showing the pathological characteristics of chronic active gastritis. Bacteriological examination showed that all Hp infections were positive in the mucosa of the esophagus, gastric body, gastric antrum, duodenum, small intestine, and large intestine. Warthin-Starry silver staining showed a large number of rods on the gastric body and gastric antrum mucosa. Hp-shaped or S-shaped Hp bacteria are stained black, and Hp accumulation can be seen in some lesions, and Hp that is clearly stained dark brown can also be seen in esophageal mucosa and duodenal mucosa.

　　(3) Comparative medicine A large number of epidemiological data confirm that Hp is the main cause of non-erosive gastritis, including chronic atrophic gastritis. Hp is a spiral, gram-negative microorganism that can cause most non-erosive gastritis and its complications. Humans infected with the bacteria 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 this regard, researchers have been trying to use various methods to infect different animals with Hp in recent years, intending to establish a human-like animal model of chronic atrophic gastritis induced by Hp infection, and set the Luo Shuang standard. This standard requires: ①Hp colonization can be seen on the gastric mucosa, and pathological changes similar to humans appear in the gastric body and antrum. ②There is a certain amount of Hp per gram of stomach tissue. ③Hp and gastric mucosa adhesion should be observed. ④ Long-term and persistent bacterial infections are required. ⑤The strain used must be a stable strain that can infect animals after a certain number of subcultures in vitro. According to this standard, the researchers successively infected with Hp and successfully induced animal models of chronic gastritis in mice, rats, Mongolian gerbils, dogs and pigs. But for most animals, the low colonization rate of Hp and the short duration of infection are always insurmountable technical obstacles that affect the success rate of their modeling. Therefore, it is its technology to select Hp susceptible animals and select Hp strains with strong motivation. The essential. For example, Hp (SS1) is a strain that is isolated and suitable for colonization in the stomach of mice through continuous passage in the stomach of mice. The strain is extremely dynamic, contains cagA and vagA, and belongs to type I Hp. Epidemiological investigations show that type I Hp is closely related to the occurrence of peptic ulcer. It contains Pathogenicity Island, expresses vacA, and has a positive vacuolar toxicity. Inoculation of this strain into the stomach of SPF-grade BABA/c mice can cause severe pathological changes in the animal's stomach tissue, including the disappearance of normal gastric glands, erosion and ulcers, etc. The success rate of modeling is 100%. Compared with mice, Hp infection is difficult to occur in the absence of gastric mucosal damage. Generally, the rat gastric mucosa is damaged first, and then Hp is used to infect it. Therefore, Hp is rarely used to prepare rats. Model of related chronic gastritis. The infection rate of MG itself to Hp is very low, but subcutaneous injection of indomethacin 20mg/kg body weight or intragastric administration of 50% ethanol 5ml/kg body weight can increase the infection rate to more than 70% and cause an obvious gastritis model. The histomorphological manifestations are: mucosal erosions within 3 weeks, neutrophil and monocyte infiltration in the mucosal layer, lymphoid follicles in the lamina propria and submucosa at 6 weeks, atrophy, intestinal metaplasia and intestinal metaplasia at 12-24 weeks ulcer. It is worth mentioning that Hp can be colonized in the stomach continuously for a long time after infection with MG, mainly in the mucus layer on the gastric surface, and a few adhere to the surface of the surface mucus cells. The amount of bacteria in the MG stomach can reach 100,000 CFU, and the lesions caused are mainly in the antrum, similar to humans. Compared with Luo Shuang's standard, except ③ which is not very typical, the others all meet the standard. In addition to the success of Hp infection in the above-mentioned animals, the replication of Hp infection models with Xisheng dogs, Xisheng pigs, and specific pathogen-free pigs has also been successful abroad. Because pigs are omnivorous animals, the anatomical structure, physiological functions and eating habits of pigs are very similar to humans, so there is a reasonable side to choosing pigs as experimental animals. However, due to the complex replication technology of the model, high environmental requirements, and expensive model building, there are few practical applications. In this context, some researchers have tried to establish Hp infection under the conditions of Xisheng, and then raise the animals under ordinary conditions. As a result, Hp can continue to exist, which solves the inevitable encounter in the whole experiment of Xisheng pig. Bottlenecks such as unaffordable technology and price. Experiments have shown that after infection with Hp, model animals first cause transient and incoherent neutrophil infiltration in the non-glandular part of the cardia, followed by lymphocyte infiltration in the glandular part of the stomach; lymphocytes start only in the submucosa It gathers with the mucosa and then develops to the submucosa and lamina propria to form discontinuous lymphoid follicles. Warthin-Starry silver staining of gastric mucosal tissue found that Hp is located between the gastric mucosal epithelium and the superficial mucus protective layer, which is similar to the performance of Hp in the human stomach. The key technology for replicating the Hp model of China No. 1 minipig is mainly to pretreat the non-steroidal drug indomethacin before Hp vaccination. Since the gastrointestinal tract, especially the gastric mucosa, contains a large amount of prostaglandins (PG), 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 mucosa The barrier function. Indomethacin can inhibit prostaglandin (PG) synthesis in the body. When indomethacin is taken into the body, the content of PG in the body, especially in the gastric mucosa, is significantly reduced, which directly damages the barrier function of the gastric mucosa and reduces the protective function of epithelial cells, thereby creating a place for Hp to settle in the gastric mucosa. The right conditions.