在HepG2细胞中虫草素的降血脂作用机制研究

he study is to confirm the silencing effiency of the vector in human hepat ocellular liver carcinoma cell line (HepG2), and observe effects of AMPKγ silencing on the AMPK situmulating activity and lipid synthesis of CCS, a natural product with known AMPK activating function. The downregulating efficacy of siRNAs on AMPKγ expression was confirmed in our previous study. The double stranded shRNA oligo was ligated to lentivirus vector and verified by sequencing. The lentiviral which can effectively inhibited protein expression levels of AMPKγ were select by western blot, and the regulation of CCS on protein expression of AMPKγ and p-AMPK in AMPKγ silence cells were detected by western blot analysis. The lipid accumulation in cells was observed by Oil-Red O stain and cells were collected for the estimation of cholesterol(TC), triglyceride(TG). The results showed that the lentiviral vector carrying an shRNA targeting the AMPKγ gene was successfully constructed. Western blotting analysis confirmed that GR085 had the highest interfering efficiency. Treatment with CCS can significantly increased the levels of phospho-AMPK in normal cells, and the level of TC,TG was reduced, but in AMPKγ silence cells the effects of CCS on AMPK activation and lipid synthesis almost completely abolished without changing the expression levels of total AMPK or AMPK γ protein. In conclusion, the AMPKγ gene may be related with AMPK activation and intracellular lipids regulaton by CCS.