Construction of recombinant adeno-associated virus vector expressing human tissue kallikrein gene and its effect on human umbilical vein endothelial cells
- VernacularTitle:激肽释放酶腺相关病毒载体的构建及对脐静脉内皮细胞功能的影响
- Author:
Peng-Li ZHU
;
Jing-Ming RUAN
;
Zhao-Xi YU
;
Hui CHEN
;
Ti-Yuan LI
;
- Publication Type:Journal Article
- Keywords:
Kallikrein;
Dependovirus;
Endothelium;
vascular
- From:
Chinese Journal of Geriatrics
2003;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct the recombinant adeno-associated virus vector(rAAV) expressing the human tissue kallikrein(HK)gene and to detect the expression of interested gene in human umbilical vein endothelial cells(HUVEC)which were infected with different titers of rAAV. Methods The HK gene was cloned into the pAAV-MCS and co-transfected AAV-293 cells with other two plasmids(the pAAV-RC,and pHelper)by lipofectamine.The recombinant AAV(rAAV HK)particles was harvested and its viral titer was measured.HUVEC were infected with different titers of rAAV-HK particles.Seventy-two hours later,the expression of the interested gene was detected by RT-PCR and ELISA.Results The AAV expression system of human tissue kallikrein gene was successfully constructed.The viral titer of recombinant AAV reached 6.2?10~7 particles/ml. When compared with the control group,the transcription of HK gene in the group which was infected with rAAV-HK increased significantly[(0.59?0.12)vs(0.26?0.05)(P＜0.05)],and the expression of HK in the HUVEC was three times more than that in the control[(120.1?40.9)vs (30.8?12.8)](P＜0.001).The transcription of eNOSmRNA in the infected HUVEC was higher than that of the control[(1.19?0.28)vs(0.66?0.11)](P＜0.05).The transcription of caspase-3 mRNA was lower than that of the control[(0.30?0.25)vs(0.67?0.27)](P＜0.05).And there was no obvious variation happened in the transcription of VEGF,ET-1,TR-B,bradykinin B1 receptor and Bradykinin B2 receptor.Conclusions When co-transfected AAV-293 cells with three plasmids (pAAV-HK,pAAV-RC,pHelper),the HK gene expression is significantly and stably increased. Introducing HK gene into HUVEC can improve endothelial transfection efficiency.
