动物造模,药效评价,肝硬化 z-bio 2024-08-22 536

　　(1) Method of replication: Adult rats were anesthetized and underwent a midline incision on the navel. The common bile duct was sutured with 7-0 silk thread at a distance of 1cm from the porta hepatis. After the surgery, regular feeding for 2-8 weeks. During this period, pay attention to observing the general activity and physical signs of the animals, and dynamically collect blood from the orbit to prepare serum for the detection of alanine aminotransferase (ALT), total bilirubin (TBIL), alkaline phosphatase (ALP), and hyaluronic acid (HA) content. At the end of the modeling process, the liver was harvested and weighed. After sectioning, a portion of the tissue was taken to prepare a homogenate for the determination of hydroxyproline (Hyp), total protein (TP), malondialdehyde (MDA), and superoxide dismutase (SOD) content. Some tissues were fixed in 10% formaldehyde solution, embedded in paraffin, and stained with HE or Masson. Some tissues were fixed with 4% glutaraldehyde and 1% osmium tetroxide, embedded with 618 epoxy resin, and ultra-thin sectioning mechanism was used to prepare sections. Uranium lead double staining was performed, and tissue morphology was examined under light and electron microscopy.

　　(2) After 5 weeks of modeling, the wet weight and coefficient of liver and spleen in the model animals significantly increased, while serum ALT, TBIL, ALP, liver tissue Hyp, MDA increased, and liver tissue TP and SOD decreased. According to the light microscope, scattered degeneration, necrosis, and inflammatory cell infiltration gradually appeared in the liver tissue of the model animal 1-2 days after surgery, which became very obvious at 3 days. At the same time as the above changes, small bile duct like epithelial cells appeared around the lobules in the portal area, gradually expanding, extending, and deepening into the lobules over time, resulting in the lobules forming a floral ring shape. At 35 days, small bile ducts and accompanying fibroblasts, collagen fibers, and capillaries form membranous septa, which connect, surround, and divide with adjacent proliferative septa until they form pseudo lobules. Electron microscopy showed that on postoperative days 1 and 2, there were scattered atrophy and degeneration of single or 2-3 liver cells around the lobules, with nuclear condensation, dense cytoplasm, and degeneration of organelles. On day 3, there was dissolution necrosis, and inflammatory cell infiltration and phagocytic cell aggregation were observed. At the same time as liver cell degeneration and necrosis, small bile duct like epithelial cells appeared and proliferated around the lobules, and the basal side of their tubular structures had a basement membrane and collagen fiber layer. At 1-2 weeks, the small bile duct like epithelial cells further proliferate and gradually extend from the periphery of the lobules along the hepatic cell line towards the interior of the lobules. After 3 weeks, significant differentiation and evolution of small bile duct like epithelial cells were observed, with most resembling differentiation into small bile ducts. The epithelial cells became cuboidal or flattened, with an increase in intermediate filaments in the cytoplasm and irregular shapes formed by concave nuclear membranes. Heterochromatin aggregated under the nuclear membrane, the lumen expanded, and microvilli sparsely distributed. The proliferation of collagen fibers around the small bile duct gradually forms a clear layer of collagen fibers. The proliferating small bile ducts, along with their accompanying phagocytic cells, mast cells, inflammatory cells, fibroblasts, capillaries, and proliferating collagen fibers, form the septa between liver cells. A small number of small bile duct like epithelial cells can also differentiate and evolve towards liver cells, while transition cells and liver cells in the form of tubular vesicles can be seen. It often wraps around a clear layer of collagen fibers, forming small liver cell nodules. At 8-12 weeks, typical collagen fiber proliferation and liver cell nodules were observed.

　　(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, resulting in ischemia and necrosis of liver cells, ultimately leading to biliary fibrosis and cirrhosis. The use of common bile duct ligation can completely obstruct the bile ducts of model animals, hinder bile excretion, and cause accumulation in the bile duct, inducing the production of free radicals in the liver, causing lipid peroxidation of cell membranes (increased MDA and decreased SOD), leading to structural damage, increased membrane permeability, and imbalanced cellular metabolism. Lipid peroxidation triggers a chain reaction, further exacerbating liver cell damage and necrosis (TP decrease). The ischemia and necrosis of a large number of liver cells induce the appearance of small bile duct like epithelial cells. The latter may belong to a type of stem cell with multi differentiation potential that proliferates and has both proliferation and differentiation functions. In the case of liver damage and necrosis of liver parenchymal cells, small bile duct like epithelial cells differentiate and evolve into tubular or nodular liver cells, while synthesizing collagen to participate in the proliferation of collagen fibers (Hyp increase), accelerating the formation and development of cirrhosis. This model can simulate the process and results of liver fibrosis and cirrhosis caused by long-term cholestasis in humans well, and the modeling method is simple, the cycle is moderate, the repeatability is good, and the success rate is high. At present, bile cirrhosis models induced by common bile duct ligation in animals such as rabbits, dogs, and monkeys, except for rats, have been successfully established and widely used both domestically and internationally.