外源性Apoptin蛋白过表达对裸鼠乳腺癌MCF-7移植瘤凋亡活性的影响及机制

Effect of exogenous Apoptin over-expression on apoptosis of orthotopic transplantation tumor of human breast cagcer cell line MCF-7 and the mechanism

目的 构建表达重组凋亡素（VP3）基因的重组逆转录病毒,探讨其对人乳腺癌MCF-7裸鼠移植瘤模型的凋亡诱导活性及机制.方法 克隆VP3基因,重组法构成逆转录病毒载体pLP-LNCX-VP3（pLLVP3）,转染PT67细胞进行病毒包装;雌激素皮贴刺激法建立去卵巢裸鼠乳腺癌皮下移植瘤模型,观察pLLVP3对肿瘤的生长抑制情况,TUNEL法检测肿瘤凋亡,Western blot印迹法检测VP3、Caspase-3和bcl-2蛋白表达.结果 重组载体pLLVP3鉴定正确,滴度为1×10^7 pfu/ml;裸鼠实验pLLVP3组抑瘤率分别为65.52%和68.23%,显著高于对照组（t=4.06,P〈0.01）,48 h可见VP3蛋白高表达.TUNEL见各组凋亡指数无差异（t1=1.05,t2=0.84,均为P〉0.05）.VP3蛋白高表达组Caspase-3表达亦升高,但bcl-2未见差异.结论 VP3基因能够诱导人乳腺癌细胞MCF-7的凋亡,可能是激活Caspase-3而发生作用.

Objective By constructing recombinant expression plasmid pLLYP3 expressing recombinant Apoptin （VP3） gene,to discuss the effect of VP3 inducing tumor cells into apoptosis on human breast cancer cell MCF-7 orthotopic transplantation tumors and the mechanism.Methods VP3 gene was cloned into the plasmid pLP-LNCX to form the recombinant plasmid pLLVP3,and MCF-7 orthotopic transplantation tumor model Was established,then pLLVP3 was infected into models.The expression of VP3 gene was detected by Western blot.TdT-mediated duTp nick end labeling （TUNEL） assay was used to verify apoptosis of tnnlor cells.At last Caspase-3 and B cell lymphoma-2 （bcl-2） were detected by Western blot.Results Sequence analysis revealed the recombination of plasmid pLLVP3,and 1×10^7 pfu/ml virus was obtained.The findings of animal experiment showed that tulnor inhibition rate of pLVP3 with low and high doses was 65.52% and 68.23% respectively,evidently higher than that in control group（t=4.06,P〈0.01）.Besides,Apoptin protein Was over-expressed.Forty-eight h after infection,TUNEL analysis showed obvious apoptosis peaks with the highest percentage rate of apoptotic cells present and cellular Caspase-3 expression could be detected in pLLVP3-infected group.Conclusion VP3 gene could induce apoptosis in human colon cancer cell line MCF-7 in vivo by activating Caspase-3 expression.