The Role Of TFAM In The Process Of Protecting Mitochondrial And Hepatic Function By Recombinant Human Augmenter Of Liver Regeneration In Rats With Obstructive Jaundice

Objectives: To study the change of mitochondrial and hepatic function in rats with obstructive jaundice after RNA interference of target gene and to explore the role of gene TFAM in the process of protecting mitochondrial and hepatic function by rhALR,and finally,to provide assistance for the clinic application of rhALR.Methods:1,We designed 4 siRNAs targeting gene TFAM and constructed the lentivirus-siRNA vector system.2,Lentiviral vector was generated by co-transfecting 293T cell with pGC-LV recombinant plasmid, pHelper1.0 and pHelper2.0 plasmids,and then the titer of LV was determined on 293T cell.3,To test the efficacy of designed siRNAs in knocking down TFAM expression,we transfected HSC-T6 cell(a rat liver stellate cell line)with lentivirus carrying siRNA or control vectors.According to the results of RT-PCR and Western blot analysis, we picked out the most effective siRNA and it was going to be used to downregulate the expression of target gene in vivo.4,One hundred and eighty Wistar rats were randomized into five groups,namely groupâ… (sham operation),groupâ…¡(OJ+RBF+rhALR),groupâ…¢(OJ+RBF+rhALR+LV),groupâ…£(OJ+RBF+rhALR+control vector) and groupâ…¤(OJ+RBF+rhALR+NaCl).Samples were collected on the 4th,7th,14th,18th,21st and 28th day after the first operation.5,The efficacy of RNAi in vivo was determined by Real time PCR and Western blot analysis.6,We observed the pathological change of liver from each group and detected the changes of hepatic function and mitochondrial function. Results:1,The results of sequencing showed that designed siRNAs were successfully integrated into the lentivirus-siRNA vector system.2,Titers of lenti-target1#, lenti-target2#, lenti-target3# and lenti-target4# were 8E+7 TU/ml, 7E+7 TU/ml, 9E+7 TU/ml, and 1E+8 TU/ml respectively.3,The result of RNAi of target gene in vitro showed that lenti-target3# was the most effective one,and the result of Western blot analysis also proved its potency.4,After bile duct ligation,the livers in all groups except groupâ… exhibited various degrees of pathological changes,and groupâ…¢showed the most obvious pathological change among all groups.With the restoration of bile flow,the pathological changes abated gradually,and the restoration in groupâ…¢was slowest among all groups.5,After bile duct ligation,there were various degrees of damage to hepatic function and mitochondrial function in groupâ…¡, groupâ…¢, groupâ…£and groupâ…¤(P<0.05,compared with groupâ… ),and the damage to groupâ…¢was more severe than those to other groups(P<0.05,compared with groupâ…¡, groupâ…£and groupâ…¤). After the restoration of bile flow,mitochondrial function and hepatic function of all groups recovered gradually,but the recovery speed in groupâ…¢was slower than those of other groups(P<0.05,compared with groupâ…¡, groupâ…£and groupâ…¤).6,The result of RNAi in vivo showed that target gene expression in gourpâ…¢decreased obviously in mRNA and protein levels and the difference among groupâ…¡, groupâ…£and groupâ…¤was not statistically significant.Conclusion:1,In this study,we successfully construced a lentiviral-siRNA vector system. The results of RNAi in vitro and in vivo proved its efficacy in knocking down target gene expression without obvious"off-target"effect and toxic reaction.2,Target gene TFAM may played a crucial role in the process of protecting mitochondrial and hepatic function by rhALR. We hypothesized that rhALR could induce the expression of TFAM, protect and repair damaged mtDNA, increase its replication number in hepatocyte, and as a result, improve mitochondrial and hepatic function,and finally promote the recovery of hepatic function after restoration of bile flow in rats with obstructive jaundice.