旁观者效应在肝癌基因治疗中的作用

ROLE OF A "BYSTANDER EFFECT" IN GENE THERAPY ON HEPATOCARCINOMA WITH HSV-tk/ACV SYSTEM

目的 :观察HSV -tk/ACV(HerpesSimplexVirusthymidinekinasegene/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 -772 1细胞为SMMC -tk细胞。SMMC -TK细胞与SMMC - 772 1细胞以不同百分比混合 ,用ACV(10 0ug/ml)培养基培养 ,MTT法检测ACV对各组细胞抑制率 (InhibitRate ,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 % ,2 0 % ,30 % ,4 0 % ,5 0 % ,6 0 % ,70 % ,80 % ,90 % ,10 0 %各组IR/St比值分别为2 .15 ,1.91,1.83,1.4 7,1.30 ,1.11,1.0 2 ,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 % ,2 0 % ,30 % ,4 0 %组最强 ,80 %～ 10 0 %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。

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.