Research On The Role Of Extracellular Proteinase Inhibitor Expi In Rat Liver Regeneration

Proteinase inhibitor gene expi has the protease activity, and involved in cell migration and apoptosis. According to the expression profile of rat regenerating liver detected with rat genome 230 2.0 chip, expi was observed to begin to express at 6h after 2/3 hepatectomy and increased consistently at 6-72h, reaching peak (33 times higher than control) at 30h. Liver regeneration, an important life process, is also involved in protein degradation, cell migration, and apoptosis and so on. Based on the above description, the study on the function of this gene in liver regeneration will be helpful in revealing the molecular mechanism of liver regeneration. However, at present, the function of this gene on liver regeneration has not been researched yet. Therefore, this paper employed the technique of hydrodynamics-based transgene to explore the influence of expi in the process of rat liver regeneration by means of adding (or interfering) this gene.Based on the nucleotide sequence of Expi, primers were designed to amplify this gene. At the same time, the two RNA interference fragments of this gene were designed. Then, 3 sets of the recombinant vectors were established by ligation with the above right fragments, respectively, which includes the expression vector pEGFP-N1-expi for gene addition assay, the interference vectors pGenesil-1.0-E6 and pGenesil-1.0-E147 for gene inference test, and the test vectors pGenesil-1.0-E6-expi, pGenesil-1.0-E147 -expi and pGenesil-1.0-HK-expi for testing whether the two interference fragments have interference effect on the target gene or not.The constructed test vectors were transgened based on hydrodynamics. At 6, 12, 24, 36, 48, 60, 72 and 84h after transgene, the interference effect of interference fragments on the target site were determined by observing whether the GFP-positive cell rates (that's transfection rate) were decreased more obviously than control. The optimum time for transgene after partial hepatectomy was obtained by statistically measuring the peak time of the hydrodynamics expression of different vectors (including expression vectors and interference vectors) in rat livers, at the same time, expi expression in rat regenerating livers from the chip data must to be considered. Finally, the transgenic-liver samples at 72, 120, and 168h after partial hepatectomy were taken to analyze the mortality, expression kinetics, index of liver regeneration, and morphology structure, etc. by using fluorescence microscopy techniques, statistical methods and conventional histological technique.DNA sequencing and electrophoresis showed: the cloned expi sequence is right; the vector was successfully constructed; the two interference fragments exhibit the more pronounced interference effect. The best time for transgene was 18 hours after partial hepatectomy.Liver index test results show that if adding expi, liver index was higher than control; if interfering expi, lower than control. In short, this study successfully cloned expi and successfully constructed the expression vector, interference vector and test vector. According to the in vivo assays, it is presumed that expi has effect to a extent on rat liver regeneration, which provides the valuable information for elucidating the molecular mechanism of liver regeneration.