Livin siRNA重组腺病毒的构建及其对K562细胞增殖、凋亡和耐药的影响

Construction of recombinant adenovirus vector of Livin siRNA and its effects on proliferation,apoptosis and drug resistance of K562 cells

目的通过改良的重组腺病毒介导方法研究Livin基因与K562细胞增殖、凋亡及耐药的关系。方法用基因工程技术将靶向Livin siRNA片段克隆至穿梭质粒pSES-HUS中,然后与骨架质粒pAdeasy同源重组,将重组成功的质粒用脂质体介导的方法转染HEK293细胞,乒乓感染收获高滴度的重组腺病毒Ad5-Livin siRNA,并将其感染K562细胞,采用Real-time PCR、Western blot检测Livin基因的干扰效果,用MTT和流式细胞仪检测其与K562细胞增殖、凋亡及多药耐药的关系。结果成功构建重组腺病毒Ad5-Livin siRNA,并感染K562细胞,在mRNA和蛋白2个水平均抑制Livin的表达。证实通过重组腺病毒沉默Livin基因,可抑制K562细胞增殖,促进凋亡,增加其对阿霉素、长春新碱和依托泊苷的敏感性。结论用重组腺病毒介导的方法抑制K562细胞中Livin基因的表达,可增加K562细胞的凋亡及对抗癌药物的敏感性。Livin可能成为治疗白血病新的分子靶点。

Objective To study the effect of recombinant adenovirus vector-mediated Livin siRNA on the proliferation, apoptosis and drug resistance in K562 cells. Methods The targeting Livin siRNA was subcloned into the shuttle plasmid pSES-HUS, and then recombinant it with adenovirus skeleton plasmid pAdeasy by homologous recombination. We transfected the product into HEK293 cells via liposome and generated recombinant adenovirus AdS-Livin siRNA. K562 cells were infected with AdS-Livin siRNA. The gene silencing efficiency of recombinant adenovirus Ad5-Livin siRNA was monitored by real-time PCR and Western blot analysis in K562 cells. The relationship between Livin siRNA and K562 cells was detected by MTT assays and flow cytometry. Results The recombinant adenovius vector of AdS-Livin siRNA was successfully constructed and infected into K562 cells. Recombinant adenovirus vector silenced Livin gene at the level of mRNA and protein, inhibited proliferation and enhanced apoptosis and sensitivity to Adriamycin, Vincristine and Etoposide in K562 cells. Conclusion The recombinant adenovirus AdS-Livin siRNA effectively silences the expression of Livin gene and enhances the apoptosis and sensitivity to anticancer drugs in K562 cells. Livin is a new molecular target of leukemia＇ s therapy.