动物造模,肝纤维化模型 z-bio 2021-11-29 878

　　(1) Method of reproduction: 4 mg/head of human serum albumin was subcutaneously injected into female Wistar rats weighing 120 to 150 g at the same dose on the 1, 15, 25 and 35 days, and human serum albumin was injected Into normal saline. Then emulsify with an equal amount of incomplete Freund's adjuvant. Ten days after the last immunization, blood was drawn for antibody testing, patients with positive anti-albumin antibodies were selected, and albumin was injected into the tail vein twice a week. Among them, 2.5 mg/mouse was injected intravenously in the first week, and then the dose of each challenge injection was increased by 0.5 mg/mouse to 4.5 mg/mouse, and the dose was maintained for at least 2 months; or the first and third times . The time was 8 mg/mouse, and the rest were 4 mg/mouse, 9 times in total. At the same time, prostaglandin E1 (PGE1) 0.2 mg/hour was subcutaneously injected twice a day before the challenge injection until the modeling was completed. In addition, each pig was intraperitoneally injected with 0.5 ml pig serum or 2 ml fresh sterile adult mixed pigs per pig twice a week for 10 consecutive weeks, and continued observation for 10 weeks after the injection was stopped. During the modeling period, the overall condition of the animals was observed daily. Weigh once a week. After the immune challenge and the subsequent observation period, whole blood was collected at different times to prepare serum, and liver was collected to prepare tissue homogenate for immune complex and biochemical examination. According to the requirements of optical and electron microscopy, the material was sampled and fixed , Embedding, sectioning, serial paraffin sections were stained with HE, VG and toluidine blue, and histomorphological examination was performed. Frozen sections are used for immunofluorescence for the detection of IgG and complement C3 in the liver.

　　(2) Model characteristics: 10 weeks after subcutaneous injection of 2.5-4.5 mg of human serum albumin, about 80% of model animals may have fibrosis and even different degrees of liver cirrhosis. Yes, the mortality rate is 20% to 30%. .. After 30 days of 4-8 mg challenge injection and PGE1 subcutaneous injection protection, model animals can develop liver cirrhosis with a mortality rate of only 5%. In the early stage of the stimulation injection, the tissue structure of the liver is almost normal, but the proliferation of fat storage cells is active. During the injection, the serum C3 content was significantly reduced. In the later period, it is the liver. The junctional cells deform and die, inflammatory cells infiltrate, the portal vein area and interlobular fibrous tissue proliferate, the liver collagen content increases, the liver sinusoids, blood vessel walls, connective tissue and filament spheroids are stained, and the serum C3 content gradually increases. Restore the state and exceed the normal value. Swine serum was injected continuously for 7 weeks. At 10 weeks after injection, the concentrations of hyaluronic acid (HA), laminin (LN) and type III collagen (PCⅢ) increased in serum and tissue homogenate of model animals. Expansion, fibers increase and extend to the lobules, connect to the adjacent portal vein area and hepatic vein, divide and surround the liver parenchyma, increase the portal vein area and septal matrix components, reduce the cell composition, and form the fibrous portal of the main extracellular matrix) deposition The portal vein cells are mostly thin, dark-stained fibrocytes; within 15 weeks of modeling, the liver tissue structure is basically the same as that of 10 weeks; portal vein area and diaphragm; within 20 weeks of modeling, liver tissue Some animals will reduce or disappear intermittently in certain fiber intervals. In addition to the aforementioned spindle-shaped mesenchymal cells, infiltration of acidic granulocytes (Eos) can also be seen in the portal vein and compartments. The continuous challenge injection peaked at 8 weeks and gradually decreased after 10 days. week. Even after 20 weeks, you can still see a small amount of Eos in the portal area. After eight consecutive weeks of injection, immunofluorescence was not only positive for IgG and C3 in the walls of blood vessels and sinuses, but also positive for the interstitium distributed in numerous filaments and codes. At the end of the 10th and 15th weeks, IgG and C3 were positive. C3 is scattered in the interstitium and portal interstitium, and its positiveness is further enhanced. At the end of the 20th week, IgG and C3 in the interstitium and sinus wall were weakened. Continuously challenge fresh pig aseptic mixed blood and inject for 10 weeks. The fibrous membrane of the model animal is formed and becomes relatively thin. At 10-14 weeks, the fibrous septum gradually thickens, the liver tissue is severely separated, and more pseudo-lobules are formed, and the area is small. This trend of change lasted for as long as 20 weeks.

　　(3) Comparative Medicine Human liver fibrosis is a clinical syndrome with complex etiology, and it is a necessary stage for the development of chronic hepatitis into cirrhosis. Immune factor-induced liver fibrosis is the main pathological feature, especially in various types of viral hepatitis and certain liver diseases. Human or heterologous animal serum enters the human body, and albumin and macromolecular substances are used as heterologous antigens to stimulate the human body to produce corresponding antibodies. When an antigen re-enters the body, it forms an antigen-antibody immune complex (IC). It mainly exists in the liver and is deposited around the portal vein and central vein, causing local inflammation, stimulating the proliferation of fat-storing cells, transforming into fibroblasts, and then secreting collagen fibers and causing fibrosis of liver tissue. At the same time, heterologous serum triggers mast cell-mediated type I allergy (LPR), causing a large amount of Eos infiltration and releasing a variety of substances, such as transforming growth factor β1 (TGF-β1). become. Primitive mesenchymal cells (PMC) and hepatic stellate (HSC) activate, induce and synthesize large amounts of ECM. This can lead to excessive ECM deposition in the tissues, which can lead to liver fibrosis. The main feature of this model is the stable pathological morphology of liver fibrosis in model animals. After the injection was stopped, the natural reabsorption of the previously formed liver fibrous tissue was relatively light. You can maintain the form of liver fibrosis. Lipid storage and fibroblasts participate in the transformation process and cause little damage to liver cells. This model is different from post-hepatitis liver fibrosis and liver cirrhosis in terms of liver cell damage, but in view of the fact that the current hepatitis virus cannot replicate to the experimental liver fibrosis model, this model is caused by immune damage. Therefore, it is a more feasible method to comprehensively study and evaluate the etiology of liver fibrosis by combining the CCl4-induced liver fibrosis animal model for chronic liver disease mainly caused by viral hepatitis. Some scholars have systematically studied the rat liver fibrosis model induced by human serum albumin, the modeling, the modeling environment, and the experimental methods used to prevent animal immune states that lead to excessive immune responses in animals, and found that death has an effect on the success rate of modeling. The impact is very important. influences. It seems appropriate to choose 6-week-old rats for model replication under environmental conditions of (22±1)°C, and daily subcutaneous injection of PGE1 is a key technology. In addition to human and pig serum, bovine serum albumin can also be used to replicate animal models of immune liver fibrosis.