K562白血病细胞中Musashi2干扰慢病毒载体的构建和鉴定

Construction and identification of Musashi2 RNA interference lentiviral vector in K562 leukemia cells

目的:构建干扰Musashi 2 (Msi2)的慢病毒载体,并探究其在K562白血病细胞株中的干扰效率。方法:将两个Msi2干扰片段分别连接入GV418慢病毒载体,与pHelper1. 0、pHelper2. 0辅助载体共转染入293T细胞进行慢病毒包装。提取细胞上清感染白血病K562细胞,并用嘌呤霉素药物稳筛,获取稳定感染细胞株。定量PCR和western blot分别检测K562细胞内Msi2的干扰效率。结果:与对照组比较,干扰组shMsi2-1和shMsi2-2的Msi2 mRNA水平分别降为3%和28%(P <0. 05);与对照组相比,优选出的shMsi2-1组中Msi2蛋白水平也显著下降(P <0. 05)。结论:成功构建Msi2干扰慢病毒载体并显著沉默白血病K562细胞中Msi2的表达,为后续研究Msi2在白血病中的功能和机制奠定实验基础。

Objective: To construct Musashi2(Msi2) RNA interference lentiviral vector and explore the interference efficiency in K562 leukemia cell line. Methods: Two Msi2 interference fragments were ligated into GV418 lentiviral vector, and co-transfected into 293T cells with pHelper1.0 and pHelper2.0 helper vectors for lentiviral packaging. The cell supernatant was extracted and infected with leukemia K562 cells, and puromycin was used to screen to obtain a stably infected cell strain. Quantitative PCR and western blot were used to detect the interference efficiency of Msi2 in K562 cells. Results: Compared with the control group, the Msi2 mRNA levels in the interference groups named shMsi2-1 and shMsi2-2 groups were reduced to 3% and 28%, respectively ( P <0.05). The Msi2 protein level in the selected shMsi2-1 group was also significantly decreased compared with the control group( P <0.05). Conclusions: The Msi2 RNA interference lentiviral vector was successfully constructed and the expression of Msi2 in K562 leukemia cells was silenced apparently, which laid a foundation for the subsequent study of the function and mechanism of Msi2 in leukemia.