慢病毒转染脐血CD34^+造血干细胞方法的优化

The Optimization of Method for Lentiviral Vector to Transfect CD34^+ Hematopoietic Stem Cells from Human Cord Blood

目的探讨慢病毒载体系统转染人脐血CD34+造血干细胞优化的转染方法。方法采用第二代、第三代慢病毒载体系统,通过改变病毒浓度、感染体积、感染复数(MOI)值、感染方式、感染时间和感染后培养基等各方面条件,将表达绿色荧光蛋白(GFP)基因的质粒pTRIPdU3-RNAiTALh-EF1a-GFP转染CD34+干细胞,于37℃、5%CO2的孵箱中培养14d后,在光学显微镜下进行集落计数和分类,并与未转染的CD34+干细胞进行比较,从而筛选出优化的转染方法。结果三质粒系统的第二代慢病毒载体转染率高于四质粒系统的第三代慢病毒载体。优化的转染条件为:新鲜分选的CD34+细胞于当日(0d),在含病毒滴度107 TU、Polybrene 2μg/mL的opti-MEM培养液中,细胞和病毒混合液于平底12孔板孔中,200×g离心1h,离心后继续共培养8h,再换新的病毒液200×g离心1h,共培养8h。按照含细胞因子的液体培养基∶半固体培养基=1∶1的比例配置转染后培养基,继续培养,可以获得较高感染率。感染病毒后的CD34+干细胞培养14d后,与未感染的CD34+细胞相似,可以形成各类血细胞集落。结论优化的慢病毒转染方法可以有效感染CD34+造血干细胞,而不影响干细胞的分化功能。

Objective To identify the best transfect conditions for lentiviral vector to transfect CD34＋ stem cells from human cord blood. Methods CD34＋ hematopoietic stem ceils from human cord blood were transduced with pTRIPdU3-RNAiTALh-EFla-GFP plasmid expressing GFP by the second generation and third generation lentiviral vector system. The transfect conditions such as the concentration of the virus, polybrene, transfect volume and media, multiplicity of infection （MOI） values, incubating time and centrifugation in 12-well plate at 200 × g were tested to obtain optimal transfect conditions. The number of CFU were counted and the types of CFU were identified by light microscope after the transfected ceils （non-infected stem cells served as control） were cultured for 14 days at a 37℃, 5M CO2 incubator. Results The second-generation lentiviral vector plasmid had higher infect rate than the third-generation. The optimal transfect conditions were determined as： fresh sorting CD34＋ cells, 107 TU virus concentration, Polybrene 2μg/mL in opti-MEM medium, centrifuged at 200 μg for 1 h and then co- culture 8 h for cells and virus mixture in one well in flat-bottomed 12-well plate （repeated once）. Both infected and non-infected CD34＋ stem cells developed CFUs with similar numbers and types of colonies after being cultured for 14 days in the cytokine-containing 1 ： 1 liquid medium/semi-solid medium. Conclusion The identified optimal conditions can enable effective lentiviral vector transduction of CD34＋ without interrupting the differentiation potential of the hematopoietic stem cells.