The Study On An Double Suicide Gene System Mediated By Adenoviral Gene Vector Targeted Human Umbilical Vein Endothelial Cells

ObjectiveTo study the selectively killing of human umbilical vein endothelial cells (HUVEC) by a double suicide gene under the regulation of Kinase Domain Insert Containing Receptor (KDR) promoter and mediated by an adenoviral gene vector. Two recombinant Adenoviral plasmid pAdKDR-CDglyTK pAdCMV-CDglyTK were constructed in a "Two-step transformation protocol", and the two newly constructed plasmids were transfected to 293 packaging cells to grow Adenovirus. HUVEC and LoVo cells were infected with either of the two resultant recombinant Adenovirus (AdKDR-CDglyTK and AdCMV-CDglyTK) respectively, the infection rate were measured with the aid of GFP expression. Infected cells were cultured in culture mediums containing different concentration of GCV and 5-FC,and the killing effects were measured.Methods1. Recombinant adenovirus constructionKDR promoter sequence CD gene sequence and TK gene sequence were generated by PCR protocol to construct pKDR-CDglyTK. Then pAdKDR-CDglyTK were generated with Adeasy ?1 system in a "two step transformation protocol. And pAdCMV-CdglyTK were constructed in the same way. The two recombinant adenoviral plasmids were then transfected to 293 packaging cells to grow Adenovirus, which were further multiplied and purified. The resultant recombinant adenoviruses were identified by PCR protocols.2. Gene transfer and prodrug sensitive experimentHUVEC cells and LoVo cells were infected with therecombinant adenoviruses at different MOI, and infection rate were measured with the aid of GFP expression visualized by fluorescence microscopy. Transgeneic cells were treated with different concentration of GCV and/or 5-FC, cell survival rate were measured 3d latter.Results1. we constructed KDR promoter CD gene and TK gene successfully, and the resultant segment were sequenced and identified by Sangon Biotechnology (Shanghai) Co., Ltd.2. AdKDR-CdglyTK and AdCMV-CDglyTK were constructed in a "two step transformation protocol". Data indicated that the "two step transformation protocol" is a method that more convenient and economical than the traditional ones.3. HUVEC and LoVo cells were infected with the two recombinant adenoviruses: the infection rate were of no differernce, and it increasing with the increasing of the MOI of the viruses.4. HUVEC cells infected with AdKDR-CDglyTK at MOI of 100 were maintained in culture medium of different concentration of GCV and/or 5-FC for 3d: transgene cells were highly sensitive to prodrugs. Cells survival rate decreased with the increasing of concentration of prodrugs. And the two prodrugs showed similar toxicities, however, a marked decrease in cell survival was observed when GCV and 5-FC were combined (p<0.05) .5. MOI of the virus plays a role in cells killing either: combined GCV and 5-FC achieved greater extent of killing with increasing MOI, and the data of the control group ( infected cells maintained in culture medium without prodrugs) indicated that virus alone was not of much toxicity.6. HUVEC cells and LoVo cells infected with AdKDR-CDglyTK- 9 -or AdCMV-CDglyTK at MOI of 100 were maintained in culture medium of different concentration of GCV and 5-FC for 3d: Both AdCMV-CDglyTK-infected HUVEC cells andAdCMV-CDglyTK-infected LoVo cells were highly sensitive to the prodrugs, the sensitivity of these two transgeneic cells were of no much difference (p=0.518) . AdKDR-CDglyTK-infected HUVEC cells were the same highly sensitive to the prodrugs (p>0.2) , however, AdKDR-CDglyTK-infected LoVo cells were far less sensitive to the prodrugs (p<0.001) .Conclusions1. Prodrug/KDR-CDglyTK system is effective in killing HUVEC cells; its killing effect correlates to the concentration of the prodrugs and the recombinant Adenovirus's MOI, Combined using confers the two prodrugs better killing effect on the transgeneic cells.2. selective kill...