以逆转病毒介导对人脐血CD34+造血细胞进行人MDR1基因转染的实验研究

目的：探讨逆转录病毒介导的MDR1基因转导入脐血CD34^ 细胞的最佳方法，为MDR1基因转导的临床应用打基础。方法：用磷酸钙沉淀法将含有人全长MDR1cDNA的逆转录病毒载体pHaMDR1/A转到包培育细胞PA317中，建立产病毒细胞系，以人脐血中分离的CD34^ 造血干/祖细胞为靶细胞，在体外进行基因转染，转导的条件为：与含病毒的上清液共培养12天，每天更换病毒上清液，上清液中加入IL-3，IL-6和SCF三种造血生长因子（HGF），转染后用集落培养法测定对COL的耐药性，用PCR检测14-17天所形成集落的MDR1 cDNA，计算转染率，用免疫组化法检测P170的阳性程度，并观察不同时间间隔加HGF对脐血CD34^ 细胞的扩增和转染的影响。结果：脐血CD34^ 细胞转染阳性为86.4％，P170的阳性率为77.0％，77.1％的集落对6ng/ml的COL耐受，57.4％的集落对7ng/ml的COL耐受。结论：此转染系统既能有较好的转导效果，也有较好的扩增效果，有一定的临床实用价值。

Experimental Study on Retroviral-Mediated Transfer of Human MDR1 gene into CD34+ Cell from Human Cord Blood

Objective:To explore the optimal methods of retroviral-mediated transfer of human MDR1gene into CD 34+ cell from human cord blood.Methods:PA317was transfected with a retrovirus vector,pHaMDR1/A containing full length human MDR1cDNA by using calci-um phosphate precipitation and a producer cell was generated.CD 34+ cells from human cord blood was used as a target for human MDR1gene transfer.CD 34+ cells was exposed to retrovirus-containing supernatant for12days.Fifty percent of the fresh supernatant was refed daily,and IL3,IL6and SCF were added irregularly into the supernatant.Results:86.4%of colonies of the transfectant was MDR1cDNA-positive and77.1%of colonies was resistant to6ng /ml Col and77.0%of cells was P170-positive.Conclusion:The condition of transduction led to effi-cient transduction,expansion of CFC and high expression rate of P170would be compatible with a clinical application.