重组逆转录病毒双表达载体的构建及其对K562细胞增殖活性的影响

Construction of double expression retroviral vectors and its effect on phenotype of K562 cells

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 vectors targeting chronic myeloid leukemia （CML） b3a2-type mRNA and investigate its effect on the phenotype of K562 cells. Methods The eGFP coding sequence was inserted into the retroviral vector pMSCV-neo to construct pMSCV/GFP, then H1-RNA pol III-based transcription cassettes was subcloned into it to form pMSCV/GFP-H1-BCR/ABL40AS. Two control vectors pMSCV/GFP-H1-BCR/ABL40S ＆ pMSCV/GFP-H1-BCR/ABL80AS were constructed in addition. All these constructions were identified by restriction enzyme analysis and DNA sequencing. After that, the recombinant vectors were transferred into retrovirus packaging cell line PT67 by using lipofectamine2000, and G418 were used to select stable virus-producing cell lines. Viral titer was determined by infection of NIH3T3 cells sequentially. The cell-growth curve was assayed, cell apoptosis was checked with Annexin V-PE/7AAD double staining and flow cytometry analysis after 24-hour infection, the PKR phosphorylation was assayed by Western blotting. Results The plasmids were successfully constructed. Four cell lines, named as PT67-MSCV/GFP, PT67-40as, PT67-40s and PT67-80as were gained by G418 selection, and virus titers were 6.2 × 10^5, 5.6 × 10^5 , 4.6 × 10^5 and 6.0 × 10^5 CFU/ml respectively. PT67-40as suspensions could induce K562 cell apoptosis by （22.54 ± 3. 19 ） % , significantly different from PT67-MSCV/GFP or PT6740s （ P 〈 0.05 ）, except PT67- 80as. The level of phosphorylated PKR was only increased in PT6740as group and PT67-80as group （by 59.20% and 60.33% , respectively）. Conclusion The double expression retroviral vectors have been successfully constructed and it can inhibit K562 cell growth and induce cell apoptosis. Therefore, a PKR-activating dependent method may be set up as a new strategy for cancer targeting therapy.