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目的 :探讨HSV TK/GCV系统对MEC 1细胞的体外作用。方法 :用脂质体介导将含有HSV 1 TK全长cDNA的真核表达质粒G1NATK转染人黏液表皮样癌细胞系MEC 1,经G418筛选 ,挑选阳性克隆 ,用PCR及RT PCR检测目的基因的整合及mRNA转录 ;分别用MTT及细胞记数法检测TK阳性细胞对GCV的体外敏感性及旁观者效应 ;用倒置显微镜、HE染色、活细胞荧光染色及TUNNEL染色观察GCV作用下MEC 1/TK的形态学变化。结果 :PCR及RT PCR分别从G418阳性克隆中成功扩增出 40 4bp的特异性基因片段 ,将此G418抗性克隆命名为MEC 1/TK ;IC50 MEC 1/TK为 0 .7μg/ml,而MEC 1为 10 0 0 μg/ml以上 ;当将TK阳性细胞和TK阴性细胞以不同比例混合 ,TK阳性细胞只占 10 %时 ,即有 90 %的细胞被杀死 ;形态学观察表明 ,经GCV作用 ,MEC 1/TK细胞变圆、脱壁、漂浮 ,部分细胞呈现核浓缩、核碎裂等特征 ,TUNNEL染色核阳性着色细胞明显增多。结论 :HSV TK/GCV系统在体外能明显杀伤MEC 1细胞 ,对GCV的敏感性比未转染的亲本细胞提高 10 0 0倍以上 ,并显示较强的旁观者效应 ;GCV可诱导部分MEC 1/TK细胞发生凋亡
Objective: To investigate the in vitro effect of HSV TK / GCV system on MEC 1 cells. Methods: The eukaryotic expression plasmid G1NATK containing HSV 1 TK full length cDNA was transfected into human mucoepidermoid carcinoma cell line MEC 1 by lipofectamine. The positive clones were screened by G418 and the positive clones were selected. The target gene was detected by PCR and RT PCR MTT and cell counting were used to detect the in vitro sensitivity and bystander effect of TK positive cells on GCV. The expression of MEC 1 / TK morphological changes. RESULTS: A 40 4bp specific gene fragment was successfully amplified from G418 positive clones by PCR and RT PCR, respectively, and the G418 resistant clone was named MEC 1 / TK. IC50 MEC 1 / TK was 0.7 μg / ml, while MEC 1 was above 100 μg / ml. When TK-positive cells and TK-negative cells were mixed in different proportions, only 10% of TK-positive cells were killed, 90% of the cells were killed. Morphological observation showed that, GCV, MEC 1 / TK cells rounded, detached and floating, some cells showed nuclear condensation, nuclear fragmentation and other features, TUNNEL stain positive staining cells increased significantly. CONCLUSIONS: HSV TK / GCV system can obviously kill MEC 1 cells in vitro and its sensitivity to GCV is more than 100 0 times higher than that of untransfected parental cells, and shows strong bystander effect. GCV can induce partial MEC 1 / TK cells apoptosis