Effects Of Adeno-associated Virus Mediated Combined Human Kallikrein Gene Transfection On The Human Endothelial Cell

Objectives: 1,To clone the human kallikrein(HK) gene and Adeno-associated virus plasmid (AAV-HK) to become the recombined AAV vector containing HK gene (rAAV/HK) and detect its concentration. 2,To study the changes of endothelial function through nitric oxides(NO) and nitric oxide synthase(cNOS) in cultured endothelial cell(HUVEC) with rAAV/HK vector.Methods:1,To amplify the HK coding region. The PCR product and AAV-MCS were cut by Xba I and EcoR I respectively. The HK was cloned into Adeno-associated virus(AAV) plasmid to become the recombined AAV plasmid (rAAV-HK).The rAAV-HK was confirmed by PCR and sequence, then co-transfecting the helper plasmid pHelper,plasmid RC and the rAAV-HK containing the human kallikrein cDNA into the packaging cell line 293 cell. The titer of rAVV/HK was measured indirectly using the pAAV-LacZ control plasmid through β-Galactosidase Staining Kit. 2,The cultured human endothelial cell HUVEC was divided into two groups. One group were infected by different concentrations of rAAV/HK vector (6.2×103mmol/l,6.2×104mmol/l,6.2×105mmol/l), then the contents of NO and vitality of cNOS were detected by nitrate reductase respectively after 48 hours and choose the highest expression of NO and cNOS as HK group. The other group was measured at the same time as control group.Results: 1,The successful construction of rAAV/HK vector was confirmed by PCR and sequence. The titers of its virus were determined to be about 6.2×107 per ml. The expression of human tissue kallikrein gene increase by RT-PCR. 2,Compared with control group, No and cNOS of endothelial cell in HK group were increased. The contents of NO and vitality of cNOS in HK group were 40.16±7.61μmol/l and 7.71±1.25U/ml respectively, while the contents of NO and vitality of cNOS in control group were 30.82±3.07μmol/l and 5.72±1.23U/ml after the HUVEC were cultured in 48 hours; There was a significant difference in 48 hours(P<0.05).Conclusions: The rAAV/HK vector can increase the NO and NOS expression of endothelial cell and improve the function of the endothelial cell. Our work provides a theoretical warranty for the use of KKS in gene therapy of human hypertension.