【摘要】 目的将含有多药耐药基因(mdr1)的逆转录病毒载体转染人胎盘源性间充质干细胞(MSC),评价mdr1基因在MSC中的表达及耐药基因导入对MSC耐受化疗药物作用的影响。方法2005年3月至2006年5月采用percoll密度梯度离心法自吉林大学二院妇产科健康分娩供者胎盘组织中分离MSC;脂质体转染法将含mdr1基因的重组质粒导入293T包装细胞,获得的病毒上清感染MSC,RT-PCR法、流式细胞术和免疫荧光染色检测mdr1基因在MSC中的表达;罗丹明(Rh123)排泌试验检测外源基因编码产物P-糖蛋白(P-g170)功能;MTT法测定转染前后MSC对化疗药物耐受性的改变。结果mdr1基因导入MSC后稳定转染效率达80.3%,RT-PCR证实mdr1基因可于转染MSC中有效表达,转染后MSC表达P-g170的阳性细胞百分率为31.6%,对照组为0.2%;Rh123排泌试验证实P-g170具有功能活性。基因转染后MSC对多种化疗药物的耐药性明显强于未转染MSC。结论mdr1基因体外转染人胎盘源性MSC可获得高效、稳定的功能性蛋白表达,且可明显提高MSC对多种化疗药物的耐受性。更多还原

【Abstract】 Objective To transfer retroviral vector mediated multidrug resistance gene (mdr1) into mesenchymal stem cells (MSCs) derived from human placenta, and assess the expression of transfered mdr1 gene. Thes also investigate the effects of mdr1 gene transduction on the resistance of MSC to chemotherapy. Methods Human placenta-derived MSCs were isolated by a Percoll density gradient. Recombinant plasmid was transferred to the retrovirus packaging cell,293T by lipofectamine mediated gene transfer. MSCs were transfected by the retrovirus. The expression of the mdr1 gene was detected by RT-PCR 、 flow cytometry(FCM) and immunofluorescence staining. FCM analysis of the accumulation and extrusion of rhodamin 123(Rh123) was used to determine the function of P-glycoprotein 170(P-g170) encoded by mdr1, and the resistance of transfected MSC to chemotherapy was detected by MTT in vitro. Results The transfection efficiency was 80.3% after mdr1 gene was transfered to MSC. The mdr1 gene was expressed effectively in transfected group, and there were 31.6% cells expressing P-glycoprotein 170,while 0.2% in control group. The function and activity of P-g170 was confirmed by the accumulation and extrusion of Rh123, and there was a significant increase in the resistance of transfected MSC compared with untransfected MSC. Conclusion Efficient and stable expression of mdr1 gene can be obtained by transfecting MSC in vitro, which enhances the resistance of transfected MSC to chemotherapy.更多还原