疾病动物模型,1型糖尿病模型 z-bio 2021-03-17 286

　　Pigs do not have spontaneous type 1 diabetes, which must be triggered by surgery or chemicals. Pancreatectomy has many disadvantages, including surgical trauma, and is not the first choice for establishing a type 1 diabetes model. There are many reports in the literature about the establishment of type 1 diabetes after the administration of chemicals that destroy pancreatic β cells in minipigs. These studies are usually used in the research of human type 1 diabetes, including: becoming a model. Research on β cell function, drug intervention and its effects. Similarly, miniature pigs are also the most suitable model for studying the inflammatory processes and genetic similarities associated with the development of type 1 diabetes. However, because diabetes must be induced through experiments, problems related to the occurrence and development of diabetes cannot be directly studied with existing models.

　　1, Stausset et al., pancreatic surgical resection model. The surgical resection of the pancreas was compared with the use of chemicals to induce type 1 diabetes in Göttingen minipigs. Did they use male G that was 11-17 months old? Ttingen miniature pigs weighing 20-35 kg. The surgery plan is as follows. Minipigs use conventional anesthesia methods to carefully remove the longitudinal incision in the upper abdomen, the pancreatic tail and surrounding tissues, while ligating the small blood vessels in the spleen and portal vein. And cut off. Then begin to peel the pancreatic body and head from the surrounding tissues, taking care not to damage the pancreatic duodenum and duodenal feeding tube. Finally, the pancreatic duct is exposed, ligated twice and incised. Close the abdomen and suture the skin. After the animal wakes up, it is returned to the breeding room to strengthen medical treatment. TazobacEF 4 g/0.5 g was treated with prophylactic antibiotics once a day for 5 consecutive days after surgery, and 1 g Novartin (Novargin) and 3 mg x 50 mg tramal (tramadol) were used for analgesia. Feeding was started 6 to 8 days after the operation, and exogenous pancreatin (Creon, 25000IE/Tag) was administered to each feed. Observe for 28 consecutive days. Experimental results show that all surgical pigs can survive shortly after surgery and develop diabetes. This model cannot achieve the production of endogenous insulin or C-peptide because the secretory cells have been completely excised. This has a very important advantage in evaluating the effectiveness of islet cell boundary transplantation. This model also has serious shortcomings. ① Surgery has a serious impact on the dynamic balance of the organism. Diabetic piglets need to slowly undergo wound repair and other physical functions. (2) Digestion physiologically loses the pancreas and pancreatic enzyme functions required for insulin resistance, glucose and hyperglycemia; (3) In order to survive, it is necessary to completely remove pigs from the pancreas during intensive postoperative treatment. Long-term survival requires drug treatment. Such as insulin and pancreatin.

　　2, chemically induced diabetes model

　　(1) Diabetes induced by streptozotocin: streptozotocin (STZ) has selective toxicity to pancreatic β cells, relatively low toxicity to animal tissues, and a high survival rate. Drugs are used to prepare animal diabetes models. The reported models include mice, rats, rabbits, dogs, pigs, monkeys, tree rew and other animals, and the applicable dose range is 40-150 mg/kg. STZ is a white powder that is easily decomposed under light and must be stored at low temperature to prevent light. After purchase, STZ should be stored at -20°C for immediate use. By comparing the diabetic models after dissolving STZ in different solvents, the citrate buffer with pH 4.5 is considered to be an ideal solvent. After dissolving STZ, the freshly prepared citrate buffer is clear and slightly yellow. Darkening of the color or formation of bubbles will impair the stability of the reagent. The chemical composition of STZ is methyl-1-nitroso-C2-D glucose. The compound enters β cells through the GLUT2 receptor, triggers DNA strand breaks, activates the repair mechanism, reduces the cell's nicotinamide adenine dinucleotide (NAD) and ATP below physiological levels, and causes cell death. Similar to human type 1 diabetes, the pharmacological effects of STZ can cause β-cell damage, leading to a significant increase in fasting blood glucose and a decrease in glucose-stimulated insulin secretion. Studies have shown that pigs are less sensitive to STZ, and the dose (35-40 mg/kg) that can successfully induce diabetes in rats does not significantly affect the glucose tolerance of miniature pigs. A dose of 85 mg/kg can cause pigs to develop diabetes and recover within 2 weeks. STZ doses of 100-150 mg/kg can cause diabetes in domestic pigs and miniature pigs. Although it is reported that G at 7-8 months old? 150 mg/kg STZ does not induce diabetes in Tingen minipigs, but 200 mg/kg STZ is successful. This fluctuation in response may be due to age differences (in rats, young animals are found to be less sensitive than older animals), or gender differences (in rats, female animals are less sensitive than male animals. I know) It may be due to reasons.

　　Some scholars have used two low-dose regimens (60 mg/kg, 8 days later: 30 mg/kg) to induce diabetes in Hanford miniature pigs and reduce their ability to secrete insulin. Among them, 4/7 animals have stable diabetes. Yucatan minipigs took 55 mg/kg and 50 mg/kg every 8 days, and similar results were obtained, leading to severe diabetes and significant weight loss. The observation period (6 months) showed persistent hyperglycemia. Some scholars also used Yorkshire pigs to inject 50 mg/kg of STZ intravenously for 3 consecutive days, which caused stable and severe diabetes.

　　In our laboratory, we discussed the impact of STZ on mini pigs in our country. I developed a single intravenous dose of 120mg/kg or 150mg/kg STZ for 5-7 months old Bama minipigs, Guizhou minipigs and Gojiyama minipigs. Using 120 mg/kg STZ resulted in 5/ 9 Diabetic animals. Four of them had transient diabetes, with symptoms of hyperglycemia ranging from 2 days to 1 month, and 1 of them had stable diabetes. Symptoms of hyperglycemia lasted for 3 months (end of experiment). Fasting blood sugar levels are the highest. All animals are less than 20 mmol/L. Using 150 mg/kg of STZ, 7/9 of the animals developed diabetes, 5/9 of them had severe diabetes, and the hyperglycemia symptoms remained at 20-35 mmol/L. The experiment ended (lasting 3 months) ). 2/9 patients had transient diabetes. The symptoms of hyperglycemia lasted for 10 to 20 days, the maximum fasting blood glucose was less than 20 mmol/L, and 2/9 had no diabetes. In diabetic minipigs administered intravenously with STZ, blood glucose changes in 4 periods: (1) a short period of hyperglycemia 2-4 hours after STZ administration, usually 20 mmol/L or lower; (2) hypoglycemia period , STZ 6-18 hours after administration, this is due to the destruction of β cells in the pancreas and the release of large amounts of insulin into the blood. During this period, the animal will die due to hypoglycemia. ③During the period of abnormal blood sugar, 20 to 48 hours after application. For ZTZ, the animal's blood glucose level is 4-25 mmol/L. (4) During the 48-hour period of persistent hyperglycemia after ZTZ administration, the animals showed persistent hyperglycemia. Therefore, we concluded that the sensitivity of Chinese miniature pigs to STZ is similar to that of other breeds of miniature pigs in the world, and at least 150 mg/kg STZ is required to induce a stable diabetes model. The susceptibility of individuals to ZTZ varies greatly, and the reason may be related to genetic variation. Due to the low production of miniature pigs in my country, inbreeding and genetic isolation are inevitable, resulting in genetic variation of large individuals. the difference. Of course, it may also be related to animal health. We found that chronic pneumonia is common in our minipig population.

　　(2) STZ induces mild hyperglycemia: Due to factors such as breed, ancestry, age and laboratory conditions, STZ is widely used to induce diabetes in piglets, but the degree of diabetes induced by STZ variability is so large that it limits the model The predictability of the effect. Nicotine (NIA) can protect β cells from a sharp drop in NAD and ATP levels by inhibiting the DNA repair mechanism associated with STZ damage. Larsen et al. As reported in rats, use male G? Ttingen minipigs studied whether nicotine (NIA) has a protective effect on STZ-induced β cell damage. 0, 20 mg/kg, 67 mg/kg, 100 mg/kg, 150 mg/kg and 230 were applied respectively, and then 125 mg/kg STZ was applied to study the phenomenon of induced impaired glucose tolerance in adult Gottingen miniature dogs. pig. A reliable method for (or) mild insulin deficiency diabetes. The results showed that adult G? who received STZ alone at a dose of 100-125 mg/kg? Ttingen minipigs can cause severe diabetes, but the severity of this disease varies from person to person. That is due to the steep slope of the dose response. Separate STZ curve. The dose window leading to mild insulin deficiency diabetes is very narrow. Similar to its effect in rats, NIA has a protective effect on pancreatic β cells in miniature pigs. The combination of IA and STZ induces diabetes in miniature pigs. Due to the influence of NIA, the dose-response curve between the obtained dose and fasting blood glucose is much slower. The use of 67 mg/kg IA and 125 mg/kg STZ can provide moderate fasting or postprandial hyperglycemia. This mild diabetes can be maintained for at least 2 months, making it a suitable model for the study of new therapeutic agents. diabetes.

　　(3) Alloxan-induced diabetes: Alloxan (ALX) is another compound that causes β-cell damage. Its mechanism of action is similar to STZ, and its diabetic effects are also very similar. ALX is also used to induce diabetes in Yucatan minipigs, Göttingen minipigs and Sinclair minipigs. A dose of 100-200 mg/kg can cause severe diabetes. ALX at a dose of 80 mg/kg can induce mild diabetes in Gottingen minipigs, and some β-cell loss is observed, but fasting blood glucose is normal. Boullion et al. established a mini-pig diabetes model in Yucatan by intravenous injection of ALX (175 mg/kg). Hainsworth et al. used this model to detect microvascular disease after 20 weeks of diabetes and to assess changes in retinal microvascular. Compared with non-diabetic pigs, the basement membrane of retinal capillaries in diabetic pigs is thicker. This change seems to be unrelated to the abnormal blood lipids caused by high-fat diet. This indicates that the model can be used to study the pathological mechanism of diabetic retina and evaluate the effectiveness of early intervention.