Effection Of The Foreign Human Kallikrein Gene On The Human Umbilical Vein Endothelial Cell And Compared With Enalapril

Objective: 1.To construct recombinant adeno-associated virus(rAAV) vectorcarring the human kallikrein(KK) gene and detect its titer. 2.The human umbilicalvein endothelial cell(HUVEC) was cultured in vitro and KK gene was transfered intoHUVEC the by viral infection form.To observe the effection of the KK gene's on thelevel of nitric oxide(NO) and endothelin-1(ET-1) and expressions of endothelialnitricoxide synthase(eNOS) gene, and to compare with enalapril.Methods: 1.The KK gene was directional cloned into the pAAV-MCS. Thisrecombinant pAAV expression plasmid was called pAAV-KK. 2. To co-transfectAAV-293 cell with three plasmids(pAAV-KK or pAAV-LacZ, pAAV-RC andpHelper) by lipofectamine2000 and pack rAAV vector carring the KK gene or LacZgene, then harvest rAAV-KK and rAAV-LacZ virus stocks. HT-1080 cell wasinfected by rAAV-LacZ with different folds of filuted virus stocks and then wasstained by in situ β-Galactosidase Staining Kit to estimate the titer of virus. 3.HT-1080 cell was passaged and stained after infected by rAAV-LacZ with the bestfolds of filuted virus stocks, observing the expression of LacZ gene in HT-1080 cell.4. The cultured HUVEC in vtro was infected by rAAV-KK with the best folds offiluted virus stocks. The level of NO, ET-1 and expressions of eNOS gene weredetected and compared with enalapril.Results: 1.The rAAV-KK and rAAV-LacZ were successfully construsted. 2. Theviral titer of the rAVV-KK was 6. 2 ×10~7IU/ mL. 3. LacZ gene could be stablyexpressed in the passaged HT-1080 cell which was infected by rAAV-LacZ. 4. Thelevel of NO(KK group: 21.29±1.465μmol/L, enapapril group: 18.24±0.836μmol/L,blank group: 16.42±1.028μmol/L, LacZ control group: 16.47±1.251μmol/L)and theexpression of eNOS gene ( KK group: 0.42329±0.038, enapapril group:0.40657±0.033, blank group: 0.35329±0.031, LacZ control group: 0.36488±0.049)in KK infected group and enapapril group increased significantly compared withcontrol group and blank group(P<0.01), while the level of ET-1 decreased(KKgroup: 199.48±16.99 pg/ml, enapapril grou: 220.29±12.18 pg/ml, blank group:262.91±17.85pg/ml, LacZ control group: 263.57±15.86 pg/ml; P<0.01 ) ; andcompared with other groups, the level of NO in KK infected group was highest; theeffection of KK gene was more effective than enalapril. Conclusion: 1.KK gene was cloned and which sequence was correct. Toconstruct the recombinant pAAV-KK plasmid vector. 2. The rAAV-KK wassuccessfully construscted and packed. LacZ gene, as a foreign gene, could be stablyexpressed in the passaged HT-1080 cell which was infected by rAAV-LacZ. 3.HUVEC was infected by rAAV-KK with 6.2×103IU/ml in vitro. The level of NO andthe expressions of eNOS gene increased and the level of ET-1 decreased. Thefunction of vascular endothelial cell was improved. That effection of KK gene wasmore effective than enalapril in increasing the level of NO. This result would providethe objective evidence for gene therapy of human vascular endothelial cellabnormality.