旁观者效应在肝癌基因治疗中的作用

目的：观察HSV－tk/ACV(Herpes Simplex Virus thymidine kinase gene/Acyclovir）系统基因治疗恶性肿瘤过程中旁观者效应的作用。方法：HSV－tk基因重组逆转录病毒载体转染PA317细胞为PA317－tk细胞。培养PA317－tk细胞即可获得有感染力的携带HSV－tk基因的病毒颗粒,然后转染肝癌细胞株SMMC－7721细胞为SMMC－tk细胞。SMMC－TK细胞与SMMC－7721细胞以不同百分比混合,用ACV（100ug/ml）培养基培养,MTT法检测ACV对各组细胞抑制率（Inhibit Rate,IR),根据各组IR与该组SMMC－tk细胞数的百分比值（IR／SMMC－tk%,IR/St）的大小判断旁观者效应作用的有无和强弱。结果：旁观效应试验表明,含SMMC-tk细胞10％,20％,30％,40％,50％,60％,70％,80％,90％,100％各组IR／St比值分别为2．15,1．91,1．83,1．47,1．30,1．11,1．02,0．93,0．84,0．76。10％－70％各组IR／St比值均大于1,存在明显旁观者效应,以10％,20％,30％,40％组最强,80％－100％三组IR／St比值小于1,旁观者效应不明显。结论：研究结果证实用HSV－tk/ACV系统基因治疗恶性肿瘤,适宜比例TK^ 细胞时,存在明显的旁观者效应,TK／细胞为10％时旁观者效应最强,这对于HSV－TK／ACV系统基因治疗恶性肿瘤具有重要意义。

Role of A "Bystander Effect" in Gene Therapy on Hepatocarcinoma with HSV-tk/ACV System

Objective:To observe the role of a "Bystander effect" in gene therapy on hepatocarcinoma with Hepers simples virus thymidine kinase gene/Acyclovir(HSV-tk/ACV).Method:Pa317-tk cells transfected by recombinant retrovirus with HSV-tk gene were cultured and the SMMC-7721 cells were infected with the cultured supernatant containing recombinant retrovirus. The SMMC-7721 cells transfected with HSV-tk gene were screened by G418 medium,and resistant clone were further identified with PCR for SMMC-tk cells apart of SMMC-tk cells,exposed to ACV(100ug/ml) as a test of a "Bystander Eeffect". The sensitivity of SMMC-tk and SMMC-7721 cells to ACV were examined by MTT assays in vitro.A rate of Inhibit Rate (IR)/SMMC-tk % (IR/St) were got to show "Bystander Effect".The larger the ratio of IR/St was,the more significant the "Bystander Effect"was.When a ratio of IR/St was >1,"Bystander Effect" was significant.Result: PCR results showed that HCV-tk gene was transfer red successfully to SMMC-7721 cells, the results of "Bystander Effect" test showed that IR/St of groups of SMMC-tk cells 5%, 10%, 20%,30% 50%, 60%, 70%, 80%, 90%, 100% were 0.92, 2.15, 1.91, 1.83, 1.47, 1.30, 1.02, 0.93, 0.84,0.76, respectively. The cell IR/St of the groups of 10% to 70% were <1 and all the "Bystander effect" was significant, and the groups of 10%, 20%, 30%, 40%, were especially significant.Conclusions:The results indicate that gene therapy on cancer with HSV-tk/ACV system has a significant "Bystander Effect" at a ratio of one HSV-tk+ cell to ten HSV-tk + cells.