动物造模,药效评价,肝纤维化 z-bio 2024-08-26 377

　　(1) Method of replication: Adult rats were anesthetized and their abdominal cavity was opened to locate and free the common bile duct in the descending mesentery of the duodenum. Two silk threads were threaded underneath and a "V" - shaped incision was made towards the liver. Tissue glue (TH glue) or N-butyl-2-cyanoacrylate at a dose of 0.3ml/kg body weight was injected into the proximal end using a 1ml syringe, followed by double ligation and cutting of the common bile duct. After the surgery, regular feeding for 8 weeks. During this period, attention was paid to observing the general activity and physical signs of the animals. Blood was collected from the dynamic orbit to prepare serum for alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), conjugated bilirubin (DBIL), alkaline phosphatase (ALP), gamma glutamyltranspeptidase (GGT), hyaluronic acid (HA), and laminin (LN) content detection. After modeling was completed, liver homogenates were taken to prepare hydroxyproline (Hyp) content determination, and liver tissue morphology examination was performed according to the requirements of light and electron microscopy.

　　(2) Starting from one week after surgery, the model animals showed jaundice; 2. At 4 and 8 weeks, the levels of serum ALT, AST, TBIL, DBIL, ALP, and GGT increased, and the levels of serum HA and LN, as well as liver tissue Hyp, gradually increased with the prolongation of bile duct obstruction time. Under the microscope, it can be seen that during 1-3 weeks of modeling, the structure of liver lobules is destroyed, and liver cells vary in size, some of which appear as balloon shaped changes. There is obvious infiltration of inflammatory cells in the portal area, bile duct dilation, cholestasis, proliferation, and proliferation of bile duct epithelial cells. A large number of fibrous cells appear and extend into the lobules to form fibrous strands. Most lobules have a large amount of type I and III collagen proliferation in the portal area; At 8 weeks, the proliferating bile ducts occupy most of the liver lobules, and the remaining liver cells are divided into isolated islands by the proliferating bile ducts. A large amount of collagen deposits along the proliferating bile ducts, forming finer fibrous septa. Electron microscopy examination showed that most of the liver cells in the model group animals had irregular nuclei, wrinkled or ruptured nuclear membranes, some nucleoli disappeared, a large number of organelles in the cytoplasm decreased, and various organelle breakdown products were visible. Some rough endoplasmic reticulum showed cystic swelling, with a large number of ribosomes lost, and some mitochondria swelled and disappeared. Incomplete rupture of cell membrane; The villi on the sinusoidal surface of liver cells shed, the intercellular connections became loose, and the gaps widened; Hyperplasia of bile duct cells, enlargement of lumen, deformation and edema of microvilli within the lumen, and even absence of microvilli; A large amount of collagen fiber deposition can be seen around the bile ducts and in the Dirichlet's cavity, and there is perisinusoidal capillary metaplasia.

　　(3) Comparative medicine shows that extrahepatic bile duct obstruction can cause dilation of the bile duct above the site of obstruction, bile stasis, increased pressure within the bile duct, and can also lead to dilation and rupture of intrahepatic bile ducts. Due to the compression of intrahepatic blood vessels by dilated bile ducts and bile leakage, liver cells undergo ischemia and necrosis. Fibrous tissue extends towards the bile ducts, surrounds the liver lobules, and spreads around the liver cells, leading to fibrosis and even developing into biliary liver fibrosis. The animal model of liver fibrosis induced by retrograde injection of tissue glue (TH glue) and common bile duct ligation has obvious characteristics of biliary liver fibrosis. The main characteristics of this model are that the inflammatory response of the model animals is relatively mild, the formation rate of liver fibrosis is fast, and the spontaneous reversal rate is low. Specifically, after the modeling surgery, liver cell necrosis is not significant. The serum ALT level, which reflects the degree of liver cell damage, only sharply increases on postoperative day 1 and rapidly declines thereafter. During 1-2 weeks of modeling, there is proliferation of bile ducts adjacent to the portal area of liver tissue and deposition of fibrous tissue around them; After 2 weeks, the fibrous tissue surrounding the bile duct begins to surround and separate the liver lobules, while the serum and tissue LN and HA levels, which reflect the degree of liver fibrosis, increase or remain at high levels. At 4 weeks, the Hpy content in liver tissue can increase to three times the normal value. During 4-8 weeks of modeling, a large amount of collagen was deposited around the diffusely proliferating bile ducts, and liver cells were significantly reduced compared to the proliferating bile ducts. The indicators reflecting bile stasis, TBIL, DBIL, ALP, and GGT, remained high, while serum and tissue LN and HA indicators remained at high levels. At 16 weeks, the liver tissue still showed significant fibrosis, and the fibrous septa became thicker. Compared with animal models of liver fibrosis induced by simple bile duct ligation or bile duct ligation and cutting, the latter two methods of ligation are prone to bile duct recanalization, making it difficult to determine the duration of bile duct obstruction. The liver fibrosis characteristics of model animals may weaken or disappear in a relatively short period of time. The reason why this model can maintain the liver fibrosis state of model animals for a long time is that injecting TH glue or N-butyl-2-cyanoacrylate into the bile duct during the modeling surgery is the technical key. Because through this method, complete obstruction of bile ducts, especially intrahepatic bile ducts, is technically ensured. Given that this model has a simple modeling method, short modeling period, long maintenance time, obvious pathological characteristics, and can dynamically use non-invasive serological indicators, many scholars have chosen this model for research on liver fibrosis. This model is more suitable for studying the formation mechanism of liver fibrosis and evaluating the efficacy of anti fibrotic drugs.