慢病毒介导的双自杀基因对大肠癌细胞的靶向杀伤作用

Targeted killing effect of lentivirus-mediated CD/TK suicide genes on colorectal carcinoma cells

目的:研究慢病毒介导的KDR启动子驱动CD／ TK双自杀基因系统对大肠肿瘤细胞选择性杀伤作用．方法:构建的重组质粒pLenti6/V5-D-KDR- CDglyTK及阳性对照质粒pLenti6.V5-D-GFP 在lipofectamine 2000介导下转染293FT细胞,包装扩增后获得病毒颗粒,体外感染表达 KDR的SW620细胞株和不表达KDR的LS174T 细胞株,荧光显微镜下计数确定阳性对照质粒的感染效率并以RT-PCR方法检测转基因细胞 CDglyTK的表达,然后给予不同浓度的前药 5-FC及GCV处理,观察该体系对不同细胞株的杀伤效应及其旁观者效应．结果:重组体对各细胞株的感染率相似,其感染率随慢病毒滴度的增高而递增．RT-PCR方法检测发现:除LS174T细胞外,SW620细胞有目的基因CDglyTK的表达．对于转基因的 SW620细胞,单用GCV(100 mg／L)时,其存活率为32.34％±2．42％．单用5-FC(2．0 g／L)时, 存活率为30．56％±2．14％．而联合应用两者时, SW620细胞存活率为5.36％±1．55％,LS174T 细胞存活率为95．48％±1．70％．SW620细胞对前药具有较高的敏感性,SW620与LS174T细胞株对前药敏感性有显著性差异(P<0．001), 双自杀基因的疗效优于任一单自杀基因的疗效(P<0．001)．当转基因细胞比率为40％时, 加入前药GCV和5-FC,SW620细胞存活率为11．42％±2．66％,而LS174T细胞存活率为 99．54％±2．61％,二者比较差异有显著性意义 (P<0．001),该体系旁观者效应明显．结论:慢病毒作为载体有较高的转染效率, KDR基因启动子可调控双自杀基因系统选择性杀伤表达KDR的大肠肿瘤细胞．

AIM： To investigate the killing effect of lentivirus-mediated double suicide genes under the regulation of kinase domain-containing receptor （KDR） promoter targeted on colorectal carcinoma cells. METHODS： 293FT packaging cells were transfected by the constructed plasmids pLenti6/V5- D-KDR-CDglyTK and pLenti6/V5-D-GFP. After blasticidin selection and cell cloning, the infectious viruses were generated. Then SW620 （with KDR expression） and LS174T cells （without KDR expression） were transfected with the obtained viruses by lipofectamin 2000. The transfection efficacy was evaluated by the fluorecence microscopy. The expression of CDglyTK was detected by reverse transcription-polymerase chain reaction （RT-PCR）. After treatment with 5-FC and GCV, the killing effects and bystander effect of CD/TK suicide genes on the two kinds of cell lines were assessed. RESULTS： The transfection efficacy was not significantly different between SW620 and LS174T cells, and elevated with the increase of virus titer. RT-PCR demonstrated that CDglyTK was expressed only in SW620 cells infected by pLen- ti6/V5-D-KDR-CDglyTK but not in LS147T cells. For the transfected SW620 cells, the survival rate was 32.34% ± 2.42% or 30.56% ± 2.14% when GCV （100 mg/L） or 5-FC （2.0 g/L） was used alone, respectively, and it was 5.36% ± 1.55% when GCV and 5-FC were used in combination. For the transfected LS174T cells, the survival rate was 95.48% ± 1.70% when GCV and 5-FC were used in combination. SW620 cells had a higher sensitivity to the prodrugs than LS174T cells did （P 〈 0.001）, and the effects of double suicide genes were markedly stranger than that of either single gene （P 〈 0.001）. Considerable bystander effect was also observed. When the infected cells covered a percentage of 40%, the survival rate of SW620 cells was 11.42% ＋ 2.66%, while that of LS174T cells was 99.54% ± 2.61% after treatment with GCV and 5-FC. There was significant difference between the two kinds of cells （P 〈 0.001）. CONCLUSION： Lentivirus-mediated CD/TK suicide genes driven by KDR promoter have specific killing effect on colorectal carcinoma cells with KDR expression.