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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 relationship between Livin gene and proliferation, apoptosis and drug resistance of K562 cells by modified recombinant adenovirus mediated method. Methods The targeted Livin siRNA fragment was cloned into the shuttle plasmid pSES-HUS by genetic engineering and then homologous recombined with the backbone plasmid pAdeasy. The recombinant plasmids were transfected into HEK293 cells by liposome-mediated method and the ping-pong infection was harvested High-titer recombinant adenovirus Ad5-Livin siRNA was transfected into K562 cells. The interference effect of Livin gene was detected by Real-time PCR and Western blot. The proliferation and apoptosis of K562 cells were detected by MTT and flow cytometry And multidrug resistance. Results Recombinant adenovirus Ad5-Livin siRNA was successfully constructed and was transfected into K562 cells. The expression of Livin was inhibited at both mRNA and protein levels. Confirmed that silencing Livin gene by recombinant adenovirus can inhibit K562 cell proliferation, promote apoptosis and increase its sensitivity to doxorubicin, vincristine and etoposide. Conclusion Recombinant adenovirus mediated inhibition of Livin gene expression in K562 cells can increase the apoptosis of K562 cells and its sensitivity to anti-cancer drugs. Livin may be a new molecular target for the treatment of leukemia.