The Study On The Change Of Leptin Levels In The Hepatic Ischemia-Reperfusion Injury Model And Its Recombination And Expression

Effect of Hepatic Ischemia-reperfusion Injury on Protein and mRNA Levels of Leptin in RatsObjective: To establish the hepatic ischemia-reperfusion injury model of rats, evaluate the effect of hepatic ischemia-reperfusion injury on serum Leptin levels and adipose Leptin mRNA expression in rats and discuss the possible reasons that influence the Leptin levels and the effects that caused by the change of Leptin levels.Methods: Forty-five male SD rats, whose mean weight was 220g, were divided into five groups randomly. The first group was the sham group. The rats in the second group were treated as the hepatic ischemia for 60 min then reperfusion for 60 min (I60R60). The third group was the I60R150 Group. The forth group was the I60R240 Group. The fifth group was the I60R360 Group. The study was performed on a 70% hepatic ischemia-reperfusion injury model. Serum Leptin levels were determined by a highly-sensitive murine Leptin radioimmunoassay, and the adipose Leptin mRNA expression was checked by reverse transcription-polymerase chain reaction (RT-PCR).Results: (1) Compared with the Sham Group, serum Leptin level has shown an increase trend in the ischemia for 60min and reperfusion for 60min (I60R60) group and the I60R150 group. Furthermore, a significant raise in the serum Leptin level turned up in the I60R360 group. (2) Compared with the Sham Group, the expression of adipose Leptin mRNA showed no differences in the I60R60, I60R150 and I60R240 Groups, but a decreased expression was found in the I60R360 Group.Conclusion: The 70% hepatic ischemia-reperfusion injury model of rats has been successfully established. Leptin has been shown as an acute phase reactant during the stress response of hepatic ischemia-reperfusion injury. The serum Leptin level increased during the early and middle stage of the hepatic ischemia-reperfusioninjury, while the expression of adipose Leptin mRNA decreased in the later stage of the injury.Expression, Purification and Identification for Immunological and Biological Activity of RecombinedHuman LeptinObjective: To obtain effective method of high recombined human leptin (rh-leptin) expression in E. coli and the method of purifying and renaturing the rh-leption protein, then to obtain a large amount of rh-leptin protein with immunological and biological activity for further research.Methods: The adipose Leptin CDs was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and cloned by PCR, and the product was connected with pGEM-T easy and transformed into DH5 α . After DNA sequencing, the positive colonies were picked out and amplified. The cleaved leptin fragment was cloned into pET-28a(+).Then, the recombined plasmid of leptin was transformed into BL21(DE3). DNA sequencing result showed that no mutation was found in rh-leptin' s CDS. After IPTG induction, the cell lysate was directly used for SDS-PAGE. After purification and renaturation, the immunological activity of rh-leptin was checked by Western-blot. Its biological activity was checked by reducing the weight and food intake and inhibiting the gastrointestinal motility of Km mice.Results: There was a new band of protein (> 50 % total protein) around 22 KDa on SDS-PAGE. The band was proved by Western-blot to be rh-leptin, which was highly expressed in the BL21(DE3)/ pET-28a(+) system , and the rh-leptin hasimmunological activity. Its biological activity was confirmed by reducing theweight and food intake and inhibiting the gastrointestinal motility of Km mice.Conclusion: Highly expressed rh-leptin can be obtained in our lab. Purifiedrh-leptin with immunological and biological activity may be used in furtherresearch.