Experimental Study On The Intervention Of Tetramethylpyrazine On Hepatocellular Oncogene And Gene Chip In Hepatic Fibrosis

ObjectiveThis article using the technology of gene chip, to analysis the gene genealogy in hepatocytes of hepatic fibrosis rat, then want to look for the differences in hepatocellular genes. The experiment is a study on the pathogenesy of hepatic fibrosis in the level of gene. At the same time, observing the intervention of Tetramethylpyrazine which is produce of biology fermentation extractive to liver fiborosis and hepatocellular oncogene.DrugThe crystal of Tetramethylpyrazine biology fermentation extractive is applied by aipuxiangliao company limited in shanghai. Tetramethylpyrazine 3.5g are dissolved in the 70ml mixture of CCl4,olive oil(6:1),which contein 0.05g Tetramethylpyrazine in a milliliter,conserved in 4℃.Gene chipThe 12K compelely gene chip of rat are purchased in shanghai biochip company limited.Method1. 120 healthy male SD rats are sepatated into three groups,which are normal,model and drug. The model group was induced by celiac injection of a mixture of CCl4 liquid which included CCl4, oliver oil(1:6), three times a week, totaled eight weeks. The normal was injected oliver oil, three times a week, totaled eight weeks. The grug was injected the mixture of Tetramethylpyrazine and CCl4, oliver oil(1:6). Two rats were put to death in the three groups every two weeks, then isolate the hepatocytes, the other six were prepared rat serum, two were prepared liver histological section.2. Preparation of liver histological section. Part of section use to preparate H-E staining liver histological section, others use to immunohistochemistry staining. 3. Preparation and detection of rat serum.4.Preparation of gene chip.5.Using liver function index of orrhology to detect the rat liver function of three groups; immunohistochemistry staining technique to detect the expression of p53 and c-met in model and drug group; gene chip technique to investigate the expression of p53 and c-met, and analyze the gene genealogy in hepatocytes in CCl4 induced hepatic fibrosis.6.SPSS was use.Datas were analyzed using one-way AVOVA. Dependent experiments1. Preparing of rat hepatocyte gene chip.2. The study of Tetramethylpyrazine which is produce of biology fermentation extractive interfere in liver fiborosis modle rats.3. The study of Tetramethylpyrazine which is produce of biology fermentation extractive interfere in liver fiborosis hepatocellular oncogene.4. The study of liver fiborosis hepatocellular gene genealogy of rats induced by CCl4.Results1. Tetramethylpyrazine which is produce of biology fermentation extractive can lessen liver fiborosis obviously.2. Tetramethylpyrazine which is produce of biology fermentation extractive can improve the function of liver. It is in 6-8w to produce a marked effectlessen.3. There are not immunohistochemistry staining p53 and c-met positive reaction in drug and normal groups. In 8w modle group, there are immunohistochemistry staining p53 positive reaction in liver histological section, most of these are around central veins. In 6w and 8w modle group, there are immunohistochemistry staining c-met positive reaction in liver histological section.4. Tetramethylpyrazine which is produce of biology fermentation extractive can down regulate the mRNA of p53-binding protein (LOC304798), its gene bank is BG671424.5. Tetramethylpyrazine which is produce of biology fermentation extractive can up regulate the mRNA of hepatocyte growth factor activator in 4w drug group, and down regulate the mRNA of hepatocyte growth factor activator in 6w drug group.6. There are 184 discrepant genes in 2w modle group compare with 2w normal group, 110 up regulation genes and 74 down regulation genes in these.; There are 273 discrepant genes in 4w modle group compare with 4w normal group, 129 up regulation genes and 144 down regulation genes in these; There are 361 discrepant genes in 6w modle group compare with 6w normal group, 134 up regulation genes and 227 down regulation genes in these; There are 1872 discrepant genes in 8w modle group compare with 8w normal group, 763 up regulation genes and 1109 down regulation genes in these.Conclusion1. Tetramethylpyrazine which is produce of biology fermentation extractive can lessen liver damage, delay the process of liver fiborosis. It is in 6-8w to produce a marked effectlessen.2. Tetramethylpyrazine which is produce of biology fermentation extractive can repress canceration in liver fiborosis later stage. It can down regulate the mRNA level of p53-binding protein (LOC304798), its gene bank is BG671424, then down regulate the protein expression. This can promote apoptosis effect of wild-type p53, and respress carcinogenesis of mutant-type p53.3. Tetramethylpyrazine which is produce of biology fermentation extractive can repress canceration in liver fiborosis later stage. It can repress c-met expression, but have not evidence of gene chip now.4. MAPK pathway participates pathogenesy of CCl4 induced hepatic fibrosis. CCl4 can up regulate CYP3A family genes of P450-ase genealogy.