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目的观察LV/p27kip1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p27kip1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/p27kip1慢病毒颗粒,对786-0细胞进行感染后分别进行WesternBlot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/p27kip1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p27kip1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p27kip1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/p27kip1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p27kip1表达可以阻滞肾癌细胞周期,抑制肿瘤细胞增殖,诱发凋亡。
Objective To observe the effect of LV / p27kip1 lentivirus infection on the malignant phenotype of renal carcinoma cell line 786-0 and the effect of subcutaneously transplanted tumor on zokor in order to find out the biological characteristics of p27kip1 in renal cell carcinoma. Gene therapy strategies provide experimental evidence. Methods The LV / p27kip1 lentivirus particles were packaged and purified by using the four-plasmid system. After infection of 786-0 cells, Western blotting, cell cycle analysis, cell proliferation assay and cell killing assay were performed respectively. BALB / C zebra mice were selected to construct subcutaneous xenografts. After successful injection of LV / p27kip1 lentivirus, the histopathological examination and TUNEL detection were completed after treatment. Results Western blot analysis showed that p27kip1 protein was highly expressed in 786-0 cells after infection, and the expression of p27kip1 protein increased with the increase of MOI. The cell cycle analysis showed that the exogenous p27kip1 protein was highly expressed in 786-0 cells The results showed that the cell proliferation curve showed a significant decrease of the number of cells from the 72nd hour after infection, and inhibited the cell proliferation with the increase of the MOI value. The cytotoxicity test showed that the MOI = 2, MOI = 4, MOI = 8 LV / p27kip1 infection on the cells have a significant killing effect, but the effect is no clear law; in vivo experiments show that mouse subcutaneous tumors under the action of the virus appeared obvious necrosis and apoptosis . Conclusion Increasing the expression of exogenous p27kip1 can block the cell cycle of renal carcinoma cells, inhibit the proliferation and induce the apoptosis of tumor cells.