Pilot Study Of Recombinant Adiponectin In Nonalcoholic Fatty Liver Disease

Objective: To detect the expression of recombinant AdipoQ in eukaryotic cells and attenuated Salmonella typhimurium which the vector was introduced into by electroporation, and to investigate the fatty degeneration inhibitory effect of AdipoQ to L-02 cells in vitro. To lay the foundation for the NAFLD gene therapy with AdipoQ.Methods: (1) Total mRNA was extracted from human fatty tissue and then the AdipoQ cDNA was obtained by RT-PCR. The AdipoQ gene was cloned into pEGFP-N1 vector and the pEGFP-N1-AdipoQ vector was constructed;(2)The recombinant plamid was transfected into COS-7 cells,and the expression of AdipoQ-GFP fusion protein in COS-7 cells was analyzed by RT-PCR and Western blot;(3) The plasmid of pEGFP-N1- AdipoQ was transfected into attenuated Salmonella typhimurium by electroporation and expressed;(4)Construction and expression recombination AdipoQ fusion proteinin in E.coli. by IPTG, to purify the recombination fusion protein by 6his marked Ni-TEDTM kit and analysed by Western blot. (5) Steatosis models of hepatocytes was established by adding oleic acid, then add the recombination AdipoQ fusion proteinin into steatosis models of hepatocytes, investigate the fatty degeneration inhibitory effect.Results: (1)The length of AdipoQ gene obtained was 744bp,which was correspond with the target gene;(2)The recombinant plamid of pEGFP-N1- AdipoQ was constructed successfully,and the fusion protein of AdipoQ-GFP was expressed in COS-7 cells;(3) AdipoQ could inhibit fatty degeneration in vitro.Conclusion: The use of oleic acid-induced L-02 liver cells can be successfully produced fatty liver cell culture model of change.Adiponectin can reduce the degree of hepatic steatosis ,it can be seen inhibition on the...