动物造模,药效评价,肝纤维化 z-bio 2024-09-02 965

　　(1) Method of replication: Adult male mice were subcutaneously injected with 20% CCl4 tea oil solution at a dose of 5ml/kg body weight every 5 days for 3 consecutive months. Or adult male rats were subcutaneously injected with 3ml/kg body weight of 60% CCl4 peanut oil solution, doubling the first dose twice a week for a total of 9 weeks. During the modeling period, observe the general condition of the animals daily and weigh them once a week. During the modeling process, whole blood was dynamically extracted to prepare serum for the detection of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein (TP), albumin (ALB), and hyaluronic acid (HA) content. After the modeling is completed, the animals are euthanized and organs such as liver, spleen, thymus, and adrenal gland are harvested, weighed, organ coefficients are calculated, and tissue morphology examination is performed.

　　(2) After injection of CCl4, the model animals gradually reduced their activity, became lethargic, had disheveled and dull hair, reduced their food intake, and slowed down their weight growth rate. Among them, at 30 days of modeling, half of the animals showed a small amount of fibrous tissue proliferation in their liver. At 60 days, most animals showed a small to moderate amount of fibrous tissue proliferation, and at 90 days, all animals showed moderate to large amount of fibrous tissue proliferation; The content of liver hydroxyproline also gradually increased with the prolongation of modeling time, rising at 60 days of modeling and becoming more significant at 90 days. The content of liver malondialdehyde and serum alanine transaminase increased significantly at all stages of modeling, and by 90 days of modeling, the proportion of plasma albumin decreased significantly. At one week of modeling in rats, serum ALT levels increased and inflammatory cells in the portal area increased, but no significant degeneration or necrosis of liver cells was found; At 3 weeks, while ALT levels increased, the liver began to swell and darken, with hepatic vein congestion and extensive steatosis of liver cells. Inflammatory cells began to infiltrate the liver parenchyma, and collagen fibers were concentrated in the portal area; At 5 weeks, ALT, AST, and HA levels increased simultaneously, while serum ALB levels decreased and globulin levels increased. The thymus began to atrophy, and the liver became significantly enlarged, with a hard and brittle texture, dark yellow color, and a heavy greasy sensation. Under the microscope, wrinkled liver cells, ruptured cell nuclei, and cell fragments were visible. A large number of inflammatory cells infiltrated the liver parenchyma, and collagen fibers extended from the portal area to the parenchyma; At 7 weeks, there was a significant decrease in body weight, a 3-fold increase in liver weight index, more significant increases in AST and HA levels, lower serum ALB levels, and higher levels of globulin. Along with thymic atrophy, the weight of the spleen and kidneys began to increase, and the liver became harder, more brittle, grayish yellow in color, and more greasy. All liver cells underwent varying degrees of degeneration, and about half of the cells died. Collagen fibers began to divide the liver parenchyma, and a small portion of the field of view showed fiber wrapping to form pseudolobules; At 9 weeks, the liver remained enlarged and hardened, but the degree of enlargement and increase in liver weight index slightly decreased. ALT decreased, while HA continued to rise. The weight of the spleen and kidneys increased more significantly, and most liver cells underwent degeneration and necrosis. Collagen fibers wrapped the liver tissue to form pseudo lobules.

　　(3) Comparative medicine liver fibrosis is a common reaction after liver cell necrosis or injury, and is an intermediate joint in the progression of many chronic liver diseases to cirrhosis. The formation of liver fibrosis is closely related to the release of various cytokines or lipid peroxidation products from necrotic or inflammatory cells. CCl4 is a selective hepatotoxic drug that activates into free radicals such as trichloromethyl radicals (CCl3) in the liver after entering the body. The latter can directly damage the plasma membrane, initiate lipid peroxidation, destroy the membrane structure of liver cells, and cause liver cell degeneration, necrosis, and fibrosis. The replication of liver fibrosis models through CCl4 is usually carried out in mice or rats, and the main routes of exposure are gavage, intraperitoneal injection, or subcutaneous injection. The advantage of gavage method is that CCl4 can directly reach the liver through the portal vein, and the highest level can be reached in the liver after 1.5 hours. However, the high mortality rate is its weakness that is difficult to overcome; Although the subcutaneous injection method has improved the modeling rate and is relatively less affected by interference factors, the modeling time is slower. Due to the tolerance of female rats to CCl4 and the low modeling rate, as well as the limited blood samples from mice, it is difficult to perform multiple indicator tests. Therefore, male rats are usually selected for the replication of this model. The characteristics of this model are clear mechanism, typical lesions, and easy operation, but the modeling cycle is too long, the animal mortality rate is high, and there is a certain natural recovery trend after stopping administration. For this reason, some researchers used CCl4 to replicate the model by adding 350mg/L of phenobarbital to drinking water to induce liver enzymes and increase the activity of cytochrome P450, thereby increasing the toxicity of CCl4 to the liver. In addition to subcutaneous injection of CCl4, researchers also mixed lard and cholesterol in the feed, and used 30% ethanol as a beverage to shorten the time for liver fibrosis formation in model animals.