核转染对成体骨髓源性干细胞的影响

Nucleofection effects on adult bone marrow-derived stem cells

背景:成体骨髓源性干细胞是实施细胞治疗的重要细胞来源。对成体骨髓源性干细胞的示踪,是研究其移行、分化的规律与机制并进一步阐明再生潜能、疗效的关键。目的:探讨经转染后的成体骨髓呈克隆样干细胞,在细胞表型、增殖能力、心肌分化潜能是否存在的影响。方法:应用核转染技术利用U-23程序将编码maxGFP报告基因的载体对成体骨髓呈克隆样干细胞进行转染,应用MTT法对核转染前后的生长曲线进行测定,使用3μmol/L5-氮胞苷对核转染前后成体骨髓呈克隆样干细胞进行向心肌分化诱导,并利用RT-PCR对向心肌分化的特异性标记物GATA4与MLC-2v的表达进行测定。使用成年SD大鼠心肌梗死左前降支结扎模型,对使用经maxGFP转染的成体骨髓呈克隆样干细胞施心内注射并于注射后的第2天和第7天对经注射心脏行冰冻切片并观察maxGFP在体内的表达情况。结果与结论:经核转染24h后,第47代及第119代成体骨髓呈克隆样干细胞的转染率为49.4%和43.1%,转染后5h,开始出现呈绿色荧光阳性的细胞,经核转染后仍能保持小圆球形、可贴壁、呈克隆样生长。MTT检测结果显示:核转染前后第47代及第119代均具有相似的生长曲线。核转染前第47代及119代细胞平均群体倍增时间分别为8.57,10.28h;核转染后分别为9.42,10.42h,各代前后比较差异无显著性意义(P=0.551,P=0.774)。RT-PCR检测结果显示:核转染前5-氮胞苷处理前后均表达GATA4,处理后MLC-2v条带更强;核转染后5-氮胞苷处理前后均表达GATA4,处理后MLC-2v条带更强,上述2个向心肌分化指标于转染前后变化并不明显。体内实验结果:心内注射经核转染后的成体骨髓呈克隆样干细胞,第2天及第7天可在经注射心肌中发现有少量绿色荧光阳性的细胞。提示,核转染可快速地将外源基因导入细胞,经核转染后的成体骨髓呈克隆样干细胞仍能保持其细胞形态、增殖能力及向心肌分化潜能,然而,经maxGFP基因核转染的成体骨髓呈克隆样干细胞,移植入心肌后只有少数细胞能表达经转染基因。

BACKGROUND：Adult bone marrow-derived stem cells are promising cell source in cell therapy. Tracking of adult bone marrow-derived stem cells is crucial to demonstrate the mechanism of stem cell migration and differentiation,and develop novel strategy for functional regeneration and stem cell therapy. OBJECTIVE：To explore effects of transfected adult bone marrow clonogenic stem cells （AMCSCs） on cell phenotype,proliferation and cardiac differentiation potential. METHODS：Plasmid-encoded reporter gene maxGFP was used for nucleofection of AMCSCs with U-23 program. Growth curves of AMCSCs before and after nucleofection were compared based on results of MTT assay. AMCSCs before and after nucleofection were treated with 3 μmol/L 5-azacytidine for inducing cardiac differentiation. The cardiac differentiation specific markers,GATA4 and MLC-2v,were applied to confirm cardiac differentiation by RT-PCR. The maxGFP transfected AMCSCs were conducted the intramyocardium injection into an adult Sprague-Dawley rat left anterior descending coronary artery （LAD） ligation model to trace the in vivo expression of transfected maxGFP gene. Fluorescence images of the injected heart were analyzed on days 2 and 7 postinjection. RESULTS AND CONCLUSION：At 24 hours following nucleofection,the transfection efficiencies of AMCSCs at passages 47 and 119 were 49.4% and 43.1%. On 5 hours,green fluorescence positive cells were observed. Following nucleofection,AMCSCs maintained round shape,and could adhere and show clone-shape growth. MTT assay results demonstrated that the passages 47 and 119 of AMCSCs exhibited similar growth curves before and after nucleofection. Mean population doubling time was 8.57 and 10.28 hours in passages 47 and 119 of AMCSCs prior to nucleofection,and 9.42 and 10.42 hours following nucleofection （P =0.551,P=0.774）. RT-PCR results showed that AMCSCs expressed GATA4 before and after 5-azacitidine treatment prior to nucleofection,and strongly expressed MLC-2v strip after treatment. AMCSCs expressed GATA4 prior to and following 5-azacitidine treatment after nucleofection,and strongly expressed MLC-2v after treatment. No significant difference was determined in above-mentioned indexes prior to and following nucleofection. In vivo experiment results demonstrated that a few green fluorescence positive cells were apparent in injected myocardium on days 2 and 7 following transfected AMCSCs injection. Results indicated that nucleofection is an effective and fast method for transfection of exogenous DNA into cell. The AMCSCs which are experienced with nucleofection are able to maintain their morphology,proliferation and cardiac differentiation potential. However,only a few transfected AMCSCs express the transferred gene,GFP,after intramyocardium injection.