The Effect Of PTEN On The Fatty Acid Oxidation And The VLDL Assembly In The Cultured Hepatocytes Of Newboned Calf

Employing RNA interference (RNAi) and overexpression technique, the role ofPTEN in the regulation of fatty acid oxidation and assembly and secrete of VLDLwas evaluated in the cultured hepatocytes of newboned calf. The expression of keyenzymes mRNA and protein and enzymatic activity were detected by fluorescentquantitation PCR and enzyme linked immunosorbent assay (ELISA) to research thealterations of fatty acid oxidation capability and VLDL assembly and secretion inheptocytes. The obtainde information conduces to understand the efficacy andmechanism of PTEN in fatty liver of dairy cow. The results are as follow:The vectors of overexpression and RNAi were built by adenvirus mediated. Thetwo high titer recombinated-adenvirus were obtain by amplicated in293cell. Themethod of recombinated-adenvirus transfected in the cultured hepatocytes ofnewboned calf was built.After the PTEN gene was silenced, the expressions of PTEN mRNA and proteinwere cut down. Compared with control group and negative control group, theexpression of key enzyme related to fatty acid oxidation were down-regulated．ThemRNA abundance and protein expression and enzymic activity of acyl-CoAsynthetase （ACSL） related to fatty acid activation were significantly lower（p<0.01）. The mRNA abundance and protein expression and enzymic activity ofcarnitine palmityl transferaseⅠ（CPTⅠ） and carnitine palmityl transferaseⅡ(CPTⅡ)related to fatty acid transmembrane transport were significantly lower（p<0.01）.The change of enzymic activity of CPTⅠ was the greatest in over threeenzymes. The expression of Acyl-CoA dehydrogenase(CAD),3-hydroxy acyl-CoAdehydrogenase (HADH), enoyl CoA hydratase(ECH) mRNA were also significantlylower （p<0.01）.The expression of HADH protein was significantly lower（p<0.05）,and the enzymic activity of CAD,HADH,ECH was significantly lower （p<0.05）. But the enzymic activity and expression of acyl-CoA oxidase(ACO)mRNA were significantly higher（p<0.01）in peroxisome. The expressions ofapolipoproteinB100（ApoB100）, apolipoproteinE （ApoE） and microsomaltriglyceride transfer protein （MTP）mRNA and protein, related to VLDL assemblyand secretion，were significantly lower（p<0.01）.The expression of LDLR mRNAwas significantly lower（p<0.05）.The concentration of VLDL was significantlylower in medium（p<0.05）. The content of TG was increased in hepatocytes.Compared with controls, overexpression of PTEN significantly up-regulatedACSL(p<0.01)，CPTⅠ（p<0.05），CPT Ⅱ (p<0.01)，CAD(p<0.01) and HADH（p<0.05） mRNAor protein expressions, which related to fatty acid oxidation. Theenzymic activity of CAD and HADH was significantly higher（p<0.05）. But theexpression of ECH and TH mRNA and enzymic activity did not markedlly change(p>0.05). The enzymic activity and expression of ACO mRNA were significantlylower（p<0.01）in peroxisome. At the same time, the expression of ApoB100(p<0.01), ApoE (p<0.05)and MTP (p<0.01) increased. The expression of low-densitylipoprotein receptor (LDLR) was slightly up-regulated, but no significantlydifference (p>0.05). To certify the impact of overexpressing PTEN on lipidosis inhepatocytes we detected the concentration of GT and VLDL. The concentration ofTG significantly was decreased in hepatocytes (p<0.05). The concentration of VLDLwas significantly increased in medium (p<0.05).Overexpressing or silenceing PTEN in hepatocytes can chang the fatty acidoxidation and assembly and secrete of VLDL by regulating the expression andactivity of key enzyme. At least in part, the regulation of PTEN could be a potentialapproach for the gene treatment of cow with fatty liver in the future.