| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所 === 92 === Hepatic dysfunction in sepsis is characterized by intrahepatic cholestasis. Our previous work has shown that inactivation of protein kinase C- (PKC) may play a critical role in modulating hepatic failure during late sepsis. Through suppression subtractive hybridization (SSH) technique, we also identified that two genes, 3-HSD and rBAT, are down regulated in late septic liver. 3-HSD and rBAT are the key enzymes in bile acid synthesis and metabolism. Because PKC plays a key role in the regulation of hepatocellular bile secretion during sepsis-associated cholestasis, therefore, PKC、3-HSD and rBAT may play a role in cholestasis-asscociated hepatic dysfunction during sepsis. It is of interest to explore if whether 3-HSD and rBAT expressions are regulated by PKC during late sepsis, as well as the causal relationship between bile acid accumulation and the expression of PKC、3-HSD and rBAT. In this study, in-vitro transfection technique and two kinds of animal models were used. In in vitro study, the clone-9 cells, in which the PKC gene was knocked down by antisense approach, were used to investigate whether or not the expression of 3-HSD, and rBAT genes are regulated by PKCα. In in vivo study, Cecal ligation and puncture(CLP) rats were used as a septic animal model and common bile duct ligation (CBDL) rats were used as an extra-hepatic cholestasis animal model.In this study, in-vitro transfection technique and two kinds of animal models were used. In in-vitro study, PKCα antisense was stable transfected in Clone-9 cells and the expression of 3-HSD, and rBAT were analyzed. In in-vivo study, Cecal ligation and puncture(CLP) rat was used as a septic animal model and common bile duct ligation (CBDL) rat was used as an extra-hepatic cholestasis animal model. The expressions of PKC, 3-HSD, and rBAT were observed at time course of 1, 3, 6, 9, and 18 h after CLP and CBDL operated respectively. Western blot analysis, Northern blot technique and RT-PCR analysis were used. The polyclonal antibody of 3-HSD and rBAT used in Western blot analysis were prepared by us. The results showed that (1) in vitro study, down expression of PKCα, 3-HSD and rBAT were found in stable transfected Clone-9 hepatocytes. (2) the protein expression of PKC was reduced at 9 and 18 h after CLP and no significant changes were found after CBDL. (3) both the mRNA and protein expressions of rBAT were decreased since 6 hours after CLP but not CBDL. (4) the mRNA and protein expressions of 3-HSD were decreased at 6, 9 and 18h after CLP and CBDL, respectively. These results suggested that (1) the 3-HSD and rBAT expressions in septic liver may be regulated by PKC as well as other mediators, and (2) bile acid accumulation may show a negative control of 3-HSD expression, whereas, the gene expressions of PKC and rBAT may be modulated by factors other than bile acid accumulation during sepsis. Since PKC and rBAT may play a distinctive role during sepsis-induced cholestasis, further study on gene regulation will be held in the near future.
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