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目的构建针对慢性粒细胞白血病bcr-abl b3a2型mRNA的双表达逆转录病毒载体,初步探讨其对K562细胞表型的影响。方法以逆转录病毒载体pMSCV-neo为骨架,构建eGFP及针对bcr-abl b3a2型mRNA的反义RNA双表达载体pMSCV/GFP-H1-BCR/ABL40AS,同时构建对照载体pMSCV/GFP-H1-BCR/ABL40S和pMSCV/GFP-H1-BCR/ABL80AS,酶切及测序鉴定各重组载体;以脂质体法转染各载体到PT67包装细胞后,G418筛选稳定的病毒产生细胞株,再以NIH3T3细胞测定病毒滴度并感染K562细胞,计数细胞数绘制细胞生长曲线,FCM检测细胞凋亡情况、并用Westernblot检测PKR激活情况。结果酶切及测序结果证实各重组载体构建完全正确;G418筛选到高滴度重组逆转录病毒生产细胞株:PT67-MSCV/GFP、PT67-40as、PT67-40s和PT67-80as,且PT67-40as上清感染组K562细胞生长受抑制,其24h时早期细胞凋亡率为22.54±3.19%,与除PT67-80as组外的其余各组相比有显著差异(P<0.05);PT67-40as和PT67-80as处理组细胞PKR磷酸化水平分别增高了59.20%和60.33%,2AP能抑制PT67-40as的作用。结论成功构建重组逆转录病毒双表达载体,并发现其能抑制K562细胞生长并引起细胞凋亡,通过激活PKR抗肿瘤可望成为肿瘤新的靶向治疗策略。
Objective To construct double-expression retroviral vector targeting bcr-abl b3a2 mRNA of chronic myeloid leukemia and to explore its effect on the phenotype of K562 cells. Methods The antisense RNA double expression vector pMSCV / GFP-H1-BCR / ABL40AS targeting eGFP and bcr-abl b3a2 mRNA was constructed by using the retroviral vector pMSCV-neo as the backbone and the control vector pMSCV / GFP-H1-BCR / ABL40S and pMSCV / GFP-H1-BCR / ABL80AS respectively. The recombinant plasmids were identified by restriction enzyme digestion and sequencing. The recombinant plasmids were transfected into PT67 packaging cells by lipofectamine. The stable virus-producing cell lines were screened by G418 and then transfected into NIH3T3 cells The virus titer was determined and K562 cells were infected. The cell growth curve was counted by counting the number of cells. FCM was used to detect the cell apoptosis. The activation of PKR was detected by Western blot. Results Recombinant retroviral vector was confirmed by restriction enzyme digestion and sequencing. The recombinant retroviral vector of PT67-MSCV / GFP, PT67-40as, PT67-40s and PT67-80as were screened by G418, and PT67-40as The growth of K562 cells was inhibited in the supernatant infection group, and the apoptosis rate of K562 cells in early infection group was 22.54 ± 3.19% at 24 hours, which was significantly different from other groups except PT67-80as group (P <0.05) The phosphorylation level of PKR increased by 59.20% and 60.33% in PT67-80as group, while 2AP inhibited PT67-40as. Conclusion The recombinant retroviral double expression vector was successfully constructed and found to inhibit the growth of K562 cells and induce cell apoptosis. Activation of PKR antitumor is expected to be a new targeted therapy strategy.