GST-π基因对人脐血造血干细胞抗肿瘤药物耐药性的影响

Influence of GST-π gene transfection on the resistance of human cord blood hematopoietic stem cells against antitumor drugs

目的:研究谷胱甘肽S转移酶-π(GST-π)基因转染人脐血造血干细胞对其抗肿瘤药物耐药性的影响。方法:取正常足月妊娠分娩抗凝脐血,采用磁分离法分离CD34+细胞,应用反转录病毒载体将GST-π基因转导入人脐血CD34+细胞。采用RT-PCR检测GST-π基因在被转染的CD34+细胞内的表达。采用克隆培养技术进行体外对药试验,检测在不同浓度的卡铂、4-羟基环磷酰胺(4-HC)或阿霉素(ADM)存在的条件下,CD34+细胞形成CFU-GM及BFU-E克隆的数目,判断GST-π基因转染对CD34+细胞耐药性的影响。并采用RT-PCR技术检测耐药CFU-GM克隆中GST-πmRNA的表达。结果:体外耐药试验显示,药物浓度为0时,实验组与对照组CFU-GM及BFU-E无显著性差异;当卡铂达到一定浓度时,经GST-π基因转染的CD34+细胞,CFU-GM及BFU-E克隆形成数显著高于对照组。ADM及4-HC存在条件下,实验组CFU-GM形成数也显著高于对照组,而BFU-E在两组差异无显著性。在对卡铂(3μg/ml)耐药的CFU-GM中,70％(7/10)检测到GST-πmRNA;对ADM(25 ng/ml)耐药的CFU-GM中,80％(8/10)检测到GST-πmRNA;对4-羟环磷酰胺(2.5μg/ml)耐药的CFU-GM中,80％(8/10)检测到GST-πmRNA。对照组中均未检测到GST-πmRNA。结论:GST-π基因转染人脐血造血干细胞,能增强造血干细胞对卡铂、阿霉素及4-羟基环?

Objective:To investigate the influence of GST-π gene transfection into human cord blood stem cells on their drug resistance against anti-tumor drugs.Methods:Human CD34+ cells were purified from anti-coagulated umbilical cord blood of normal full-term pregnancies by using magnetic cell sorting method.GST-π gene transfection into human cord blood CD34+ cells was carried out by using a retrovirus vector PLJ-GST-π.In vitro drug resistance test was carried out with colony assay in the pres- ence of carboplatin,4-hydroperoxycyclophosphamide(4-HC)and adriamycin individually at different con- centrations.The GST-π gene expression in drung resistant CFU-GM colonies was detected with RT-PCR. Results:Colony assay showed that there was significant difference in CFU-GM formation between GST-π gene transfected and untransfected CD34+ cells in the presence of cytotoxic drugs.RT-PCR showed that GST-π mRNA was detected in 8 of the 10 4-HC resistant CFU-GMS,8 of the 10 adriamycin resistant CFU-GMS,and 7 of the 10 carboplatin resistant CFU-GMS in transfected group,while the colonies in control group were GST-π mRNA negative.Conclusion:Transfection of GST-π gene could confer,to some extent,resistance to cord blood stem cells against carboplatin,4-HC and adriamycin in vitro.