中文摘要：

目的探讨Ki67基因小干扰RNA（Ki67．siRNA）增殖型腺病毒对肺癌细胞的示伤作用及IXlD／的表达情况。方法提取重组腺病毒的DNA，PCR鉴定正确后，大量扩增，氯化铯梯度离心纯化，测病毒滴度。ZD-Ki67感染人肺癌细胞系（A549），通过荧光显微镜下观察和结晶紫染色法检测细胞病作用、IVITT法检测细胞存活、Western印迹法检测E1A、逆转录-聚合酶链反应（RT-PCR）检测Ki67表达。结果PCR鉴定表明ZD55-Ki67包含目的基因且无野生型腺病毒的污染；ZD55-Ki67滴度为4．5×10^10PFU／ml免疫印迹法证实ZD55-Ki67在肿瘤细胞中表达E1A；ZD55-Ki67抑制Ki67表达及肺癌细胞生长的作用显著优于Ad-Ki67。结论ZD55-Ki67能够有效抑制Ki67基因的表达，降低肺癌细胞的增殖能力，为肺癌研究和抗肿瘤基因治疗提供新的策略。

英文摘要：

Objective To investigate the anti-tumor effects of replication-competent adenovirus expressing short interference RNA targeting Ki67 gene （Ki67-siRNA） and the expression level of Ki67 gene on lung cancer A549 cells in vitro. Methods The recombined adenoviruses（ZD55-Ki67）, extracted DNA, were verified by PCIL Viruses were propagated on HEK293 cells and purified by CsCI gradient according to standard techniques, and functional PFU titers were determined by plaque assay on 293 cells. The antitumor potential of ZD55-Ki67 to A549 cells was evaluated by fluorescence microscopy, MTT assay and crystal violet dye methock The expression of E1A was also detected by West-blot. The effect of ZD55- Ki67 on the Ki-67 expression of A549 cells was detected by RT-PCR. Results The analysis of PCR indicated the recombinant adenovirus ZD55-Ki67 containing Ki67-siRNA gene and without wild adenovirus. Functional PFU titers of ZD55-Ki67 were 4 × 10^10 PFU/ml. West-blot analyses indicated ZD55-Ki67 can express E1A in adenovirus-infected A549 cells. The expression of Ki67-siRNA gene delivered by ZD55- Ki67 is more effective to inhibit the proliferation and the Ki67 expression in mRNA levels of A549 cells than Ad-Ki67 in vitro. Conclusion ZD55-Ki67 can inhibit the expression of Ki67 gene and proliferation of lung cancer A549 cells. ZD55-Ki67 could be a powerful tool in further investigations of Ki67 gene and a novel therapeutic strategy for lung cancer.