SARI基因过表达对K562细胞系生物学影响的研究

发布时间：2018-06-16 02:22

本文选题：SARI + 病毒感染　；参考：《中国实验血液学杂志》2015年03期

【摘要】：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。
[Abstract]:Aim: to investigate the effect of sari gene overexpression on the biological function of K562 cells by constructing the expression vector of sali lentivirus. Methods: the target gene was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and the pLOV.CMV.GFP plasmid was recombined to construct the expression vector pLOV.CMV.GFP-SARI. Positive viral particles were obtained by sequencing, packaging and titer determination. K562 cell line was infected with viral particles. Q-PCR and Western blot were used to detect the expression of SARI gene and protein in K562 cells. The proliferation of K562 cells was detected by CCK-8 method. Apoptosis and cell cycle were analyzed by flow cytometry. Results: Sari gene was obtained by RT-PCR and the lentivirus expression vector containing sari gene was successfully constructed. The high expression of SARI gene in K562 cells was verified at molecular and protein levels, which was compared with that of blank group and Mock group infected with empty vector. In the overexpression of sari vector, the cell proliferation was significantly inhibited, the apoptosis rate was increased, but the cell cycle was not changed significantly. Conclusion the overexpression of the Sari gene can inhibit the proliferation and promote the apoptosis of K562 cells, suggesting that inducing the overexpression of the sari gene may play an important role in inhibiting the occurrence and development of CML.
【作者单位】：华中科技大学同济医学院附属协和医院检验科;华中科技大学同济医学院附属协和医院干细胞中心;
【基金】：国家自然科学基金(81100356) 湖北省自然科学基金(2014CFB466)
【分类号】：R329.2

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