目的:研究肿瘤特异性双自杀基因载体pc-ChCDTK对人视网膜母细胞瘤Y79细胞的体外杀伤作用。方法:将双自杀基因载体pc-ChCDTK电转染Y79细胞株。通过RT-PCR和Western blot方法确定CDTK在体外培养的细胞中表达情况。用HPLC法测定转染Y79细胞中5-FC向5-FU的转化效率。向载体转染和未转染的Y79细胞中加入不同浓度的前体药物5-FC(0,25,50,100,200,400,800mg/L)和/或GCV(0,2,4,8,16,32,64mg/L),5d后用MTT法检测Y79细胞的平均存活率。结果:双自杀基因载体pc-ChCDTK电转染Y79细胞成功:在载体转染的Y79细胞中,RT-PCR扩增出长度为403bp的特异条带,Western blot检测见一59kD大小的条带,与CDTK基因序列分析的预期结果一致。双自杀基因载体系统致Y79细胞存活率降低:转染Y79细胞中,5-FC向5-FU转化的效率为84.5%。转染和未转染Y79细胞中加入5-FC或GCV后平均细胞存活率均随浓度增加而降低,两组比较当5-FC浓度达到100mg/L,GCV浓度达到8mg/L后各对应浓度组间均存在差异(P<0.05)。转染组间比较,当5-FC达200mg/L,GCV达16mg/L后,5-FC+GCV组平均细胞存活率低于单加5-FC或GCV组,有统计学差异(P<0.05)。结论:双自杀基因载体pc-ChCDTK转染Y79细胞后,在Y79细胞被转录和翻译成CDTK双自杀基因蛋白。给予5-FC和GCV达到一定浓度时,它们转化成细胞毒性物质对Y79细胞产生杀伤作用。双前体药物的杀伤作用大于单前体药物。

AIM:To investigate the killing effects of tumor cells specific vector of suicide gene of CDglyTK driven by hTERT promoter on retinoblastoma Y79 cells in vitro. ·METHODS:At first,the recombinant plasmid was transfered into Y79 cells with electroblot as a delivery system. RT-PCR and Western blot analysis were used to determine the CDTK mRNA and protein expression in Y79 cells which had been transfected by pcDNA3.1-CMV-hTERT-CDTK plasmid vector. The conversion efficiency of 5-FC into 5-FU in CDglyTK-expressing Y79 cells was measured by HPLC. The killing effects of double suicide genes on Y79 cells that treated with 5-FC,GCV of different concentrations were determined by the method of MTT. ·RESULTS:The expression of CDTK gene in Y79 cells was certificated by RT-PCR and Western blot and a 59kD protein was obtaind which was equal to the sequence expection of CDTK gene. In MTT analysis,there was significant difference between transfected and non-transfected survival Y79 cells(P<0.05).The survival Y79 cells treated with 5-FC and GCV was lower than 5-FC or GCV. ·CONCLUSION:The recombinant plasmid vector pcDNA3.1-CMV-hTERT-CDTK can be transcribed and translated into CDTK fusion protein in Y79 cells.The transfer of the CDglyTK fusion gene into Y79 cells followed by the administration of 5-FC or GCV can kill Y79 cells in vitro. The killing effect of two predrugs is stronger than that of one predrug.

R739.7