四川大学学报（医学版）2013，Vol.44Issue(1)：130-134,5.
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
摘要
Abstract
Objective To identify the best transfect conditions for lentiviral vector to transfect CD34+ stem cells from human cord blood. Methods CD34+ hematopoietic stem cells 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 (MOD 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 cells (non-infected stem cells served as control) were cultured for 14 days at a 37 ℃ , 5% 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, 107TU virus concentration, Polybrene 2 μg/mL in opti-MEM medium, centrifuged at 200 ×g 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.关键词
慢病毒载体/CD34+造血干细胞/基因转染Key words
Lentiviral vector/CD34+ hematopoietic stem cells/Gene transfect引用本文复制引用
周睿卿,龚玉萍,林娟,何茜,万永凤..慢病毒转染脐血CD34+造血干细胞方法的优化[J].四川大学学报（医学版）,2013,44(1):130-134,5.基金项目
国家自然科学基金(No.30770912)、四川省科技厅社会公益项目(No.2008SZ0017)和教育部回国留学人员启动金(No.20071108-18-4)资助 （No.30770912）
