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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。
To investigate retrovirus-mediated MDR1 gene transduction into cord blood CD34 + cells the best method for the clinical application of MDR1 gene transduction lay the foundation. METHODS: The retrovirus vector pHaMDR1 / A containing human full-length MDR1 cDNA was transferred into packaging cell PA317 by calcium phosphate precipitation method to establish virus-producing cell line. CD34 + hematopoietic stem / progenitor cells isolated from human cord blood were used as target cells , In vitro gene transfection, transduction conditions: virus-containing supernatant co-cultured for 12 days, daily replacement of the virus supernatant, the supernatant was added IL-3, IL-6 and SCF three hematopoietic (HGF). After transfection, the drug resistance to COL was determined by colony culture. The MDR1 cDNA of the colonies formed 14 to 17 days after PCR was used to calculate the transfection rate. The positive rate of P170 was detected by immunohistochemistry. The effects of HGF on the expansion and transfection of cord blood CD34 + cells were observed. Results: The positive rate of transfection of cord blood CD34 + cells was 86.4%, the positive rate of P170 was 77.0%, the colonies of 77.1% were tolerant to COL of 6ng / ml, and the colonies of 57.4% were resistant to COL of 7ng / ml. Conclusion: This transfection system has better transduction effect, better amplification effect and certain clinical value.