慢病毒介导的重组p27^kip1对肾癌恶性表型影响

目的观察LV/p27^kip1慢病毒颗粒感染对肾癌细胞株786-0恶性表型影响及祼鼠皮下移植肿瘤的影响,以明确p27^kip1在肾癌中的生物学特性,期望为肾癌的预后评估及基因治疗策略提供实验依据。方法利用四质粒系统包装并纯化LV/p27^kip1慢病毒颗粒,对786-0细胞进行感染后分别进行Western Blot检测、细胞周期分析、细胞增殖测定、细胞杀伤率测定。选取BALB/C祼鼠,构建皮下移植瘤成功后注射LV/p27^kip1慢病毒,结束治疗后进行组织病理学检查及TUNEL检测。结果通过外源基因的导入,WesternBlot显示p27^kip1蛋白在感染后的786-0细胞中高表达,且跟随MOI值的增高而表达量相应增多;细胞周期分析表明外源性p27^kip1蛋白在786-0细胞高表达可抑制细胞由G1期向S期过渡;细胞增殖曲线提示感染后第72小时起细胞出现显著的数量下降,且随着MOI值的增加提高对细胞增殖的抑制;细胞杀伤性检测表明MOI=2、MOI=4、MOI=8组的LV/p27^kip1感染对细胞有显著杀伤作用,但影响效果无明确的规律;体内实验证实祼鼠皮下肿瘤在病毒的作用下出现了明显的坏死及凋亡。结论增加外源性p27^kip1表达可以阻滞肾癌细胞周期,抑制肿瘤细胞增殖,诱发凋亡。

Influence on the Malignant Phenotype of Renal Cell Carcinoma Caused by the Ectogenesis of p27^kip1 Induced by Lentivirus

Objective To observe the effect of lentivirus LV/p27^kip1 on the malignant phenotype of the 786-0 RCC cell line and the transplanted subcutaneous tumor of nude mice.The biological characteristics of p27^kip1 in RCC are identified in order to provide experimental evidence for judging the prognosis and applying gene therapy of RCC.Methods 786-0 cells were infected by lentivirus LV/p27^kip1,which was purified and packaged by using four plasmid system in advance.Experiments of the infectious cells were performed by using Western blot,flow cytometry and MTT respectively.BALB/C nude mouse which contained transplanted subcutaneous tumor was injected with lentivirus LV/p27^kip1.Histopathology and TUNEL trials were applied to examine the effect of the treatment.Results After the recombination of exogenous genes,high expression of p27^kip1 protein was noted in the infected cells by using Western blot,and the quantity of expression was increased with the increasing MOI.Cell cycle analysis indicated that the expression of exogenous p27^kip1 protein in 786-0 cells can inhibit the cell transition from G1 to S phase.Cell proliferation curve showed that the number of cells decreased markedly at the 72nd hour after infection.Cell killing activity determination indicated that the infection of LV/p27^kip1 protein at the group of MOI=2,MOI=4 and MOI=8 resulted in remarkable lethal effect on 786-0 cells.In vivo experiment proved the subcutaneous tumor of the nude mouse presented remarkable necrosis and apoptosis after the injection of LV/p27^kip1.Conclusion High expression of exogenous p27^kip1 protein can restrict the cell cycle of RCC,inhibit its proliferation and induce apoptosis.