摘要
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转到包装细胞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MDR1cDNA,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。
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.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2002年第10期689-693,共5页
Chinese Journal of Clinical Oncology
基金
国家"九五"攻关项目
卫生部科学基金项目资助
