CAJ | 학술논문

目的探讨重组逆转录病毒载体介导mdr1基因转染胎盘源性间充质干细胞(P-MSC)以应用于基因治疗的可行性、安全性.方法 Percoll密度梯度离心法自人胎盘组织中分离培养间充质干细胞(MSC),利用重复感染方法将含有mdr1基因的逆转录病毒载体导入P-MSC,经逆转录多聚酶链式反应(RT-PCR)检测mdr1 mRNA表达,荧光细胞分选法(FAGS)检测目的基因表达,同时对mdr1-MSC的干细胞特性进行鉴定及分析.结果转染细胞中显示mdrl mRNA表达；转染P-MSC表达P-gp的阳性细胞百分率为(27.6±5.1)％,明显高于非转染组的(0.4±0.1)％,两组比较差异有统计学意义(t=14.291,P＜0.01)；转染MSC在G0/G1期所占比例为95.40％,符合干细胞增殖特点.超微结构揭示其具有典型的低分化干细胞超微结构特点,且在特异诱导条件下具有向成脂、成骨定向分化潜能.结论由重组逆转录病毒介导mdr1基因体外转染胎盘源性MSC可获得高效的P-gp表达,且转染细胞可依然保持其干细胞特性.
목적 탐토중조역전록병독재체개도mdr1기인전염태반원성간충질간세포(P-MSC)이응용우기인치료적가행성、안전성.방법 Percoll밀도제도리심법자인태반조직중분리배양간충질간세포(MSC),이용중복감염방법장함유mdr1기인적역전록병독재체도입P-MSC,경역전록다취매련식반응(RT-PCR)검측mdr1 mRNA표체,형광세포분선법(FAGS)검측목적기인표체,동시대mdr1-MSC적간세포특성진행감정급분석.결과 전염세포중현시mdrl mRNA표체；전염P-MSC표체P-gp적양성세포백분솔위(27.6±5.1)％,명현고우비전염조적(0.4±0.1)％,량조비교차이유통계학의의(t=14.291,P＜0.01)；전염MSC재G0/G1기소점비례위95.40％,부합간세포증식특점.초미결구게시기구유전형적저분화간세포초미결구특점,차재특이유도조건하구유향성지、성골정향분화잠능.결론 유중조역전록병독개도mdr1기인체외전염태반원성MSC가획득고효적P-gp표체,차전염세포가의연보지기간세포특성.
Objective To explore the feasibility and safety of mdr1 gene transferred into placenta derived mesenchymal stem cells (P-MSCs) by reconstructed retroviral vector.Methods Human P-MSCs were isolated and expanded by Percoll density gradient and then transduced repeatedly by reconstructed retroviral vector containing mdr1 gene.The transfection and expression of mdr1 gene was detected by reverse transcription-polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FAGS).Meanwhile,the biological features of mdr1-MSCs were identified and analyzed.Results The expression of mdr1 mRNA was found in transfected cells.The expression of P-glycoprotein (P-gp) encoded by mdr1 gene was(27.6 ±5.1)％ in the transfected P-MSCs cells versus (0.4 ± 0.1)％ in the non-transfected P-MSCs cells (t =14.291,P ＜ 0.01).The percent of P-MSCs at quiescent phase (G0/G1 phase) was around 95.40％ and it was in accord with the characterization of stem cells.The mdr1-MSCs exhibited typical ultrastructures of low-differentiated stem cells.Moreover,they still retained the potency of adipogenic and osteogenic differentiation in the presence of appropriate conditioned media.Conclusion A stable expression of P-gp may be obtained by reconstructed retroviral-mediated transfection in vitro.And transfected MSCs retain the characteristics of stem cells.