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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。
Objective To transfect human mdr1 retrovirus vector into human placenta derived mesenchymal stem cells (MSC) to evaluate the expression of mdr1 gene in MSCs and the effect of drug resistance gene introduction on the chemoresistance of MSCs to chemotherapy Impact. Methods From March 2005 to May 2006, percoll density gradient centrifugation was used to isolate MSC from the placenta of obstetric and gynecological obstetrics and gynecology in the Second Hospital of Jilin University. The recombinant plasmid containing mdr1 gene was transfected into 293T by lipofection method Cells were infected with virus supernatant MSC, RT-PCR, flow cytometry and immunofluorescence staining mdr1 gene expression in MSC; Rhodamine (Rh123) secretion test exogenous gene encoding product P-glycoprotein (P-g170) function; MTT assay before and after transfection of MSC chemotherapy tolerance change. Results The mdr1 gene was transfected into MSCs and the transfection efficiency was 80.3%. The mdr1 gene was confirmed by RT-PCR in transfected MSCs. The percentage of positive cells expressing P-g170 was 31.6% after transfection and 0.2% in control group ; Rh123 excretion test confirmed that P-g170 has functional activity. After transfection, MSCs were significantly more resistant to various chemotherapeutic agents than non-transfected MSCs. Conclusion The mdr1 gene transfection of human placenta derived MSC in vitro can obtain efficient and stable functional protein expression, and can significantly improve the tolerance of MSCs to a variety of chemotherapeutic drugs.