The Construction Of Targeted Gene Therapy Vector And Its Cytoxicity On The Colorectal Carcinoma Cells

Objective:To construct targeted plasmid vector pcDNA3.1(-)Cp-CD-TK which contains CEA promoter.To study CD-TK expression driven by CEA specific promoter and its killing effect on CEA-positive colorectal carcinoma cells.By comparing the tumor growth inhibition effects among fusion gene and individual genes,find out whether there is co-effect;find out the optimum concentration;and observe the "bystander effect".Methods:By PCR,CEA promoter,CD and TK genes were amplified from extracted the genomic gene of human blood cells,plasmid pMD18-CD,and tgCMV/Hy-TK respectively,and then use enzyme restriction,ligation and other molecular biology method to subclone the CEA promoter,CD,and TK genes into pcDNA3.1(-) to generate pcDNA3.1(-)Cp-CD-TK.The transfection of tumor targeted plamid pcDNA3.1(-)Cp-CD-TK was liposome-mediated into the CEA-positive cells(SW480) and CEA-negative cells (Hela) respectively.The expression of CD-TK gene under the control of CEA promoter in the two kinds of cells was detected by RT-PCR.By MTT assay,the inhibition of SW480 cells and Hela cells transfected with the plasmid pcDNA3.1(-)Cp-CD-TK was detected at the presence of prodrugs 5-fluorocytosine and ganciclovir.By MTT assay,the CEA-specific killing effects of the Cp-CD-TK system on transfected cells treated with different drug scheme were detected.By mixing pcDNA3.1(-)Cp-CD-TK transfected SW480 cells and untransfected SW480 cells in a different ratio,"bystander effect" of pcDNA3.1(-)Cp-CD-TK transfected SW480 cells were observed at presence of the different concentration of drug.Results:1.PCR-generated Cp gene fragment(519bp).CD gene fragment(1310bp).and TK gene fragment(1129bp) were identified to be correctly cloned by DNA sequence analysis.2.The correct construction of targeted gene therapy vector pcDNA3.1(-)Cp-CD-TK was identified by gel electrophoresis and DNA sequence analysis and obtained.3.Combinant plasmid was transfected into the two kinds of cells.By RT-PCR, CD-TK fusion gene was proved to express in the CEA-positive cells(SW480),while silent in the CEA-negative cells(Hela).4.In cytotoxicity experiment,the SW480 cells transfected with targeted vector pcDNA3.1(-)Cp-CD-TK were sensitive to prodrug 5-Fc and GCV.In single prodrug group,GCV or 5-Fc at the concentration of 1μg/mL,160μg/mL,the cell growth inhibition ratio are 32.33%and 31.54%(P＜0.01),respectively.In combination group, cell growth inhibition was significantly higher to single prodrug,and cell growth inhibition ratio is 60.70%(P＜0.01).5.Mixed pcDNA3.1(-)Cp-CD-TK transfected SW480 cells and untransfected SW480 cells in a different ratio,it was significant that there was the "bystander effect" of pcDNA3.1(-)Cp-CD-TK transfected SW480 cells at presence of the different concentration of drug.The drug combination group shows more effective killing effect and "bystander effect" than the single prodrug group(39.28%,P＜0.01).Conclusions:Targeted gene therapy vector pcDNA3.1(-)Cp-CD-TK was constructed successfully.The expression of CD-TK fusion gene driven by CEA promoter was proved to specifically appear in the CEA-positive cells(SW480) while not in the CEA-negative cells(Hela).The cytotoxicity of the targeted gene therapy vector pcDNA3.1(-)Cp-CD-TK on the SW480 colorectal carcinoma cells could be achieved at the presence of 5-Fc and/or GCV and the most effective anti-tumor effect could be obtained when 5-Fc and GCV are administered at the same time.The double suicide gene has significant killing effect and "bystander effect" on colorectal carcinoma in vitro.The results demonstrate that a new kind of targeted gene therapy system has been constructed,which cast new light on colorectal carcinoma gene therapy.