| In this study, in vitro primary cell culture, quantitive RT-PCR, and chromatometry were applied to research the expression and activity of the key enzyme and receptor for hepatic glyconeogenesis and fat mobilization in hepatocyte and adipocyte of dairy cow. The focal point of this study is to probe the regulation mechanism of NPY on the above mentioned metabolism. The recombinate NPY and NPY-PTD were coloned and expressed by prokaryotic expression system.According to the publishing sequence in GenBank, primers were designed and RT-PCR was employed to amplify the partial cDNA of PC,PECPK, HSL,ACC, LEP, PPARα, PPARβ,GULT1 and GULT2, and the target sequences were recombinated into cloning vector, respectively.Homology of the target sequences were all above 90%.The method of fluorescent quantitation RT-PCR was established to detect the mRNA expression of PC,PECPK, HSL,ACC, LEP, PPARα, PPARβ,GULT1 and GULT2.Bovine hepatocyte was cultured by the modified two steps collagenase perfusion. The effects of NPY (0, 50 , 100 , 200 , 500 , 1000 pg/mL)on mRMA expression of PC,PECPK, PPARα, PPARβ,GULT1 and GULT2 in hepatocyte were detected by fluorescent quantitation PCR. The result showed that: the mRNA expression and activity of PEPCK and PC in hepatocytel was increased with increasing concentration of NPY in culture media. The level of PPARs mRNA in hepatocyte was increased with increasing concentration of NPY in culture media, but the level of GLUTs mRNA was decreased. The effect of 200 pg/mL NPY (0, 4, 8, 12, 16, 24h) on the above parameters was detected by fluorescent quantitation PCR too. The result showed that: the expression levels of PEPCK mRNA , PC mRNA and PPARs mRNA in liver cell were increased, the enzyme activity of PEPCK and PC was also increased, and the level of GLUTs mRNA were decreased.Bovine preadipocyte were cultured by the method of adipocyte culture reported by previous literature. The effect of NPY (0, 50 , 100 , 200 , 500 , 1000 pg/mL)on mRNA expression of HSL,ACC, LEP, PPARα, PPARβ,GULT1 and GULT2 in adipocyte were detected by fluorescent quantitation PCR. The result showed that: the expression levels of HSL mRNA in fat cell were decreased with increasing concentration of NPY in culture media. Enzyme activity of HSL was also lower than the controls. The level of ACC mRNA, LEP mRNA and PPARs mRNA in fat cell was increased with increasing concentration of NPY in culture media, but the level of GLUTs mRNA were decreased. The effect of 200 pg/mL NPY (0, 4, 8, 12, 16, 24 h) on the above parameters were detected by fluorescent quantitation PCR. The result showed that: the expression levels of ACC mRNA, LEP mRNA, and PPARs mRNA in fat cell were increased with delaying the time treated by NPY, and the activity of HSL and the expressing of HSL mRNA was decreased, the level of GLUTs mRNA were decreased.NPY(0, 50 , 100 , 200 , 500 , 1000 pg/mL)and insulin(20μmol/L) were simultaneously added in the media to detected the effect of NPY on adipocyte and hepatocyte. The result showed that the expression of HSL mRNA, PEPCK mRNA and PC mRNA were down-regulated by insulin, NPY could improve the effect of insulin on HSL mRNA in adipocyte ,and down-regulated the effect of insulin on PEPCK and PC of hepatocyte.NPY and NPY-PTD gene were cloned and the sequenced. The product of PCR was cloned into a clone vector pMD18-T. The obtained gene had no signal peptide and was ligated with an expression vector pGEX-3X to construct the prokaryotic expression plasmid Pgex-3x-NPY and Pgex-3x-NPY-PTD. The positive recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express NPY and NPY-PTD by IPTG. The specific expressed protein was detected by SDS-PAGE, and then it's biological activities was identified by angiogenesis response of chick chorioallantoic membrane (CAM).It is conclued that NPY could increase glyconeogenesis in hepatocyte and decrease fat mobilizationin adipocyte in vitro, and those effect of NPY are dose dependent and time dependence.
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