Construction Of Recombinant Adenovirus Carrying Human APM1 Gene And Its Protective Effect In Human Umbilical Endothelial Cells Treated With Palmitic Acid

OBJECTIVE:1,To construct recombinant adenovirus vector carrying human APM1 gene;2,To investigate the effects of palmitic acid(PA)on the expression of Inducible nitric oxide synthase(iNOS)protein,endothelial nitric oxide synthase (eNOS) protein and the level of Nitric oxide in cultured human umbilical endothelial cells (HUVECs )in vitro; To investigate the effects of Ad-APM1 on the expression of iNOS,eNOS protein and the level of Nitric oxide in HUVECs induced by palmitic acid.METHODS:1,Plasmid pINCY-APM1 with human adiponectin gene was considered as templete, polymerase chain reaction(PCR) was used to amplify adiponectin gene which were cloned into eukaryotic expression vector pDC315-EGFP,then the products were co-transfected into HEK293 cell line with adenovirus DNA helper plasmid pBHGlox△E1,3cre ,Ad-APM1 was obtained by site-specific recombination. The expression of adiponectin was detected by Real-time PCR and Western blot respectively. 2,Human umbilical endothelial cells (HUVECs )were cultured in vitro and exposed to PA,the cell viability was measured by MTT assay.,Nitric oxide levels was measured by Griess reagent assay, iNOS,eNOS protein were detected by Western blot.Then cells were pretreated with Ad-APM1 at the MOI of 100 for 48h prior to PA exposure.Nitric oxide levels was measured by Griess reagent assay. iNOS,eNOS protein were detected by Western blot.RESULTS:1,The recombinant adenovirus-APM1 was constructed successfully and ,The expression of adiponectin can be detected after Ad-APM1 infected HEK293 cells by Real-time PCR and Western blot. 2,HUVECs viability were significantly reduced in a time-dependent and a dose-dependant manner after exposure to PA at range of 100~800μmol/L for24~48h.The exposure of PA at the concentration of 400μmol/L to HUVECs for 24h produced the maximal response level of iNOS protein but not at higher concentration. Meanwhile ,the levels of nitrite was elevated obviously.But it may not affect the expression of eNOS protein . Cells pretreated with Ad-APM1 for 48h prior to PA exposure can significantly attenuated the elevated levels of nitrite and the expression of iNOS protein, meanwhile ,Ad-APM1 can enhanced the expression of eNOS protein (P<0.001).CONCLUSION: 1,The recombinant adenovirus carrying human APM1 gene was successfully constructed by the method of homogenous recombination in cells.2,The expression of iNOS protein and the levels of nitrite were elevated obviously in HUVECs induced by PA.But the expression of eNOS protein may have no difference among the treatment of different concentration of PA .3,Ad-APM1 may protected HUVECs from damage induced by PA via attenuated the elevated levels of nitrite and the expression of iNOS protein.