摘要
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
Objective:To estimate the effect of HSV1-TK/GCV suicide gene therapy on human salivary gland epidermoid carcinoma cell line MEC-1. Methods: Expression vector G 1NAtK containing HSV-TK cDNA was transfected into MEC-1 cells.After transfection, the cells were selected by G418 for two weeks. The integrated gene and mRNA were detected with PCR and RT-PCR. The cytotoxicity and bystander effect were estimated by MTT and typan blue exclusion assay. The morphological changes after GCV treatment were observed with HE and 33258 stain and in situ cell apoptosis detection kit. Results: The 404 bp DNA fragment was amplified through PCR and RT-PCR in the transfected cells respectively. TK positive clone was named MEC-1/TK. The sensitivity of MEC-1/TK to GCV was 1 000 times more than that of parent MEC-1 cells.More than 90% of MEC-1 cells were killed by 10 μg/ml of GCV when only 10% of MEC-/TK cells were present. The morphological changes included shrinking,detaching and floating of the cells. Some of the cells showed nucleus condensation and breakage of nucleus. A lot of cells showed nucleus positive in in situ apoptosis detection. Conclusion: HSV1-TK/GCV can confer MEC-1/TK cell killing efficiently. MEC-1/TK also has strong bystander effects. HSV1-TK/GCV system confers its effect, in part, by inducing apoptosis in TK positive cells.
出处
《实用口腔医学杂志》
CAS
CSCD
北大核心
2003年第5期458-461,共4页
Journal of Practical Stomatology
