SARI基因过表达对K562细胞系生物学影响的研究

Biological Effects of the SARI Over-expression on K562 Cell Line

目的:通过构建SARI慢病毒表达载体,探讨SARI基因过表达对K562细胞生物学功能的影响。方法:采用RT-PCR方法获得目的基因并重组p LOV.CMV.GFP质粒,构建pLOV.CMV.GFP-SARI表达载体。通过测序鉴定、病毒包装和滴度测定后,获得阳性病毒颗粒,并以病毒颗粒感染K562细胞系。采用Q-PCR及Western blot方法验证感染后K562细胞SARI基因转录和蛋白水平的表达情况,同时采用CCK-8法检测K562细胞的增殖情况,以流式细胞术分析细胞凋亡和细胞周期变化。结果:利用RT-PCR方法获得SARI基因并成功构建了含SARI基因的慢病毒表达载体,从分子及蛋白水平验证了其在感染后K562细胞中的高表达;与空白组和感染空载体的Mock组相比,过表达SARI载体组的细胞增殖显著受抑、细胞凋亡率增加,而细胞周期无显著变化。结论:SARI基因过表达可抑制K562细胞的增殖并促进其凋亡,提示诱导SARI基因过表达可能对于抑制CML发生发展具有重要的作用。

Objective： To construct a lentivirus vector carrying SARI gene and to investigate its biological effects on K562 cells. Methods：SARl was amplified from the plasmid containing SARI cDNA and subcloned into pLOV. CMV. eGFP virus vector. After sequencing, lentivirus packaging, titering, the viruses of SARI-pLOV. CMV. eGFP were harvested and tansfected into the K562 cells. Real-time quantitive PCR and Western blot were performed to validate the SARI expression at the level of mRNA and protein respectively. Simultaneously, the proliferation, apoptosis and cell cycle of K562 cells were detected by CCK-8 and flow cytometry respectively. Results： The SAR/ overexpressed lentivirus vector was successfully constructed. The mRNA and protein levels of SARI increased significantly in the pLOV. CMV. eGFP-SARI group, which was confirmed by Q-PCR and Western blot; as compared with blank and mock groups, SAR/over-expression leaded to significant proliferation inhibition and increased apoptosis of K562 cells, without visible effects on cell cycle. Conclusion： the over-expression of SAR/gene obviously suppresses the cell proliferation of the K562 cells as well as promotes the apoptosis. The results implied that the induction of the SAR/gene expression may be an important candidate therapeutic method for the CML.