DEVELOPMENT OF GENE THERAPY FOR RANAL CELL CARCINOMA

单纯疱疹病毒胸苷激酶（HSV-TK）基因转移到癌细胞后再用更昔洛韦（GCV）治疗是癌症基因治疗的重要策略。由GCV产生的GCV-三磷酸通过HSV-TK的作用有效地杀死了HSV-TK转导的细胞。此外，邻近的非转导肿瘤细胞也会被旁观者效应杀死，尽管这种效应的机制尚不完全清楚。我们提出了一个问题，是否全身免疫反应参与体内肿瘤消退。利用含有HSV-TK基因的逆转录病毒载体，在CMV启动子驱动下建立TK+肾细胞癌细胞系，并将其皮下植入BALB/c小鼠。GCV治疗TK+肿瘤细胞完全消退后，用未转导的肿瘤细胞攻击动物。观察到攻击肿瘤细胞的排斥反应或明显的生长抑制。肿瘤排斥小鼠脾细胞的^<51>Cr释放试验表明，肿瘤反应性T细胞是系统性诱导的。抗CD8抗体能有效阻断靶细胞的裂解，而抗cd4抗体则不能，说明大部分裂解是由经典CD8+ ctl介导的。为了研究肿瘤特异性ctl的诱导机制，我们检测了MHC I类分子在肿瘤细胞上的表达。ⅰ类分子在HSV-TK/GCV处理细胞上特异性升高。这些结果表明，单纯疱疹病毒- tk /GCV系统不仅具有直接杀伤细胞和旁观者效应的短期肿瘤消退作用，而且具有疫苗接种效应的长期肿瘤消退和预防复发作用。

Transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene into cancer cells followed by treatment of ganciclovir (GCV) is an important strategy of cancer gene therapy. HSV-TK transduced cells were efficiently killed with GCV-triphosphate generated from GCV by the action of HSV-TK.In addition, adjacent non-transduced tumor cells are killed by the bystander effect, althogh the mechanism of this effect is not fully understood. We addressed a question whether the systemic immune response was involved in tumor regression in vivo. TK+RENCA (renal cell carcinoma) cell lines was established using a retroviral vector containing the HSV-TK gene driven by the CMV promotor and implanted subcutaneously into BALB/c mice. After complete regression of TK+tumor cells by GCV treatment, the animals were challenged with non-transduced tumor cells.Rejection or significant growth inhibition of challenged tumor cells was observed. The ^<51>Cr release assay of the spleen cells from the tumor rejected mice demonstrated that the tumor-reactive T cells were systemically induced. The lysis of target cells was efficiently blocked by anti-CD8 antibody but not by anti-CD4 antibody, suggesting that most of the lysis was mediated by classical CD8+ CTLs. To study the mechanism of induction of the tumor specific CTLs, we examined the expression of MHC class I molecules on tumor cells., Increased levels of class I molecules were detected specifically on HSV-TK/GCV treated cells.These results suggested that HSV-TK/GCV system might be useful not only for short term tumor regression due to the direct cell killing and bystander effect but also for long term tumor regression and prevention of recurrence due to the vaccination effect.