DEVELOPMENT OF GENE THERAPY FOR RANAL CELL CARCINOMA

将单纯疱疹病毒胸苷激酶（HSV-TK）基因导入肿瘤细胞，再用更昔洛韦（GCV）治疗是肿瘤基因治疗的重要策略。GCV通过HSV-TK作用产生的GCV-三磷酸能有效地杀伤HSV-TK转导的肿瘤细胞，并能通过旁观者效应杀伤邻近的未转导的肿瘤细胞，但其机制尚不完全清楚。我们解决了一个问题，即全身免疫反应是否参与体内肿瘤消退。使用含有由CMV启动子驱动的HSV-TK基因的逆转录病毒载体建立TK+RENCA（肾细胞癌）细胞系，并皮下植入BALB/c小鼠。GCV处理TK+肿瘤细胞后，用未转导的肿瘤细胞攻击动物，观察到攻击的肿瘤细胞的排斥或明显的生长抑制。来自<51>肿瘤排斥小鼠的脾细胞的13 Cr释放测定证明肿瘤反应性T细胞是全身诱导的。抗CD 8抗体能有效地阻断靶细胞的裂解，但抗CD 4抗体不能阻断，表明大部分裂解是由经典的CD 8 + CTL介导的。为了研究肿瘤特异性CTL的诱导机制，我们检测了肿瘤细胞上MHC I类分子的表达。结果表明，HSV-TK/GCV系统不仅可通过直接杀伤细胞和旁观者效应使肿瘤细胞在短期内消退，而且可通过疫苗效应使肿瘤细胞在长期内消退和预防复发。

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.