动物造模,药效评价 z-bio 2024-09-26 799

　　(1) The model production involves cutting a fragment containing HCV 5'UTR and structural genes (C1+E1+E2) from a pHCS plasmid using enzyme digestion. After passing through an intermediate vector pMT, the fragment is further digested to construct a new plasmid (pCBE). After amplification and purification, the concentration is adjusted to around 1 μ g/L. Using microinjection method, the transgenic (pCBE) was injected into the male anterior nucleus of mouse fertilized eggs at an injection rate of 1-2 ml/egg, and then transplanted into the fallopian tubes of 6-week-old pseudo pregnant female mice. About 20 tubes were transplanted from each side of the fallopian tubes, and natural offspring were born after their development. After 4 weeks of growth, cut off 0.5-1cm long mouse tails to detect the integration of Hcv DNA in transgenic mice; Isolation and extraction of liver, kidney, heart, spleen, intestine, lungs, brain and other organs from neonatal mice in a sterile environment to detect HCV DNA transcription levels in transgenic mice.

　　(2) The high expression of HCV structural genes in early mouse development can lead to stillbirth; Moderate expression can lead to severe multi organ tissue damage; Low expression or no expression allows mice to survive, and over 80% of low expression mice can develop hepatic steatosis after 6 months of age. HCV structural genes have significant cytotoxic effects and are closely related to their expression levels. High expression of HCV structural genes can rapidly cause severe damage to multiple organs in mice, resulting in 46.7% of mice dying within 3-5 days, thus confirming the significant direct cytopathic effect of HCV structural proteins.

　　(3) Since the successful establishment of HCV transgenic mouse models in comparative medicine in the 1990s, various HCV transgenic mouse models have been developed, which not only provide important technical guarantees for studying the mechanism of HCV infection in cells, virus assembly, transport, and immunopathology in liver cells, but also provide new methods for the research and screening of anti HCV drugs. Although there have been many reports on transgenic mice as HCV infection models, there are still many problems due to the different technical methods used in various studies. There is still no consensus on whether the liver cell damage caused by HCV in transgenic mice is due to the direct action of the virus or the interaction between the virus and the body's immune system, as they are in an immune tolerant state. Therefore, it is difficult to apply HCV transgenic mice to research related vaccines.