HSV_1tk/GCV基因治疗系统的旁观者效应观察

An in Vitro Study on Bystander Effect of the HSV 1 tk/GCV Gene Therapy System

探讨逆转录病毒 (retrovirus ,RV )载体介导的单纯疱疹病毒胸苷激酶 (HSV1 tk )基因转染 ,联合抗病毒药物核苷类似物羟甲基无环鸟苷 (ganciclovir,GCV )对人卵巢上皮癌细胞系TYK细胞杀伤过程中所产生的旁观者效应。采用脂质体介导法将PLNTK5质粒转入包装细胞PA317后 ,以滴度最高的PA317病毒上清液感染TYK细胞 ,遗传霉素G418筛选后 ,得到带有HSV1 tk基因的TYK细胞。将细胞按不同比例混合培养后 ,给予 10 μg/mlGCV ,4d后用MTT法计算细胞存活率 ,观察旁观者效应。PLNTK5质粒成功转入PA317细胞 ,病毒滴度最高者为 6× 10 5cfu/ml。用病毒上清液感染TYK细胞 ,成功地得到了表达HSV1 tk的卵巢癌细胞株TYK/tk。混合培养结果显示 ,TYK/tk细胞占混合细胞 10 %时 ,低浓度的GCV就可使一半以上的细胞杀死 ,证明了HSV1 tk/GCV治疗方法存在着旁观者效应。逆转录病毒可介导HSV1 tk基因转入TYK细胞并获稳定表达 ,HSV tk/GCV系统存在旁观者效应。

To evaluate the bystander effect of retrovirus mediated herpes simplex virus thymidine kinase (HSV 1 tk) gene transference following administration of ganciclovir (GCV) on ovarian cancer cells PLNTK5 plasmid was transferred to an amphoteric packing cell line PA317 using lipofectin method,and a stable virus producing cell line was to be established Ovarian epithelial cancer cell line TYK was infected by the PA317 budded virus whose titer was the highest PCR and Southern blot were resorted to demonstrate the successful transduction and transcription of the HSV 1 tk gene TYK/tk cell were mixed with different ratio and cultured in 96 well plates After 48h co culture,10μg/ml GCV was added into half of the wells,the other half served as controls 4 days later,viability was obtained by MTT test Experimental results revealed that stable virus producing cell line was established after PLNTK5 plasmid transferred into PA317,and TYK/tk cells that express the HSV 1 tk gene were obtained successfully After mixed culturing of TYK and TYK/tk cells in different radio,when the ratio of HSV 1 tk positive cells reached 10%,the tumor cell killing proportion was almost 50% The test showed that bystander effect of TYK/tk/GCV system could improve the antitumoral efficiency It concludes that the HSV 1 tk gene can be transducted into human ovarian cancer cell line TYK under the mediation of retrovirus,and bystander effect was found in TYK/tk/GCV system