增强型绿色荧光蛋白基因电穿孔转染骨髓间充质干细胞的研究  

TRANSFECTION OF ENHENCED GREEN FLUORESCENT PROTEIN GENES INTO RAT MESENCHYMAL STEM CELLS MEDICATED BY ELECTROPORATION

作　　者：王晓军[1] 崔连群[2] 王敏[3] 刘继东[1] 李峰[1] 盖玉生[1] 

机构地区：[1]山东大学山东省立医院,250021 [2]山东大学山东省立医院心内科 [3]山东科技大学生物医学工程系,250014

出　　处：《中国组织化学与细胞化学杂志》2005年第1期16-22,共7页Chinese Journal of Histochemistry and Cytochemistry

摘　　要：　目的　建立大鼠骨髓间充质干细胞的分离、培养方法, 探讨电穿孔法介导外源基因转染骨髓间充质干细胞的可行性及转染效率。方法　Ficoll PaqueTMPlus淋巴细胞分离液分离大鼠骨髓间充质干细胞 (rMSCs) 并进行原代培养和传代扩增, 免疫组化的方法对其初步鉴定。用荧光显微镜、细胞计数法和流式细胞仪分析转染效率。结果　电穿孔法可较高效转染 rMSCs, 转染率为 (32. 8%±3)%。该条件下电转染后的MSCs其生长曲线与转染前的细胞比较无明显变化。结论　优化条件的电穿孔法具有较高的介导外源基因表达于 rMSCs的效率, 且对 rMSCs的生物学行为没有明显影响。Objective To cultivate and identify the mesenchymal stem cells(MSCs) obtained from rat bone marrow and to study the transfection efficiency of enhanced green fluorescence protein (EGFP) genes into MSCs by electroporation. Methods Rat MSCs were separated and purified by gradient centrifugation with Ficoll-Paque TMPlus. The cells were cultivated and then expanded by subculture successively. The growth curves were drawn, and the morphology observed.The immuno-phenotypes of the MSCs were evaluated with immunocytochemical methods. The pIRES2-EGFP plasmid was amplified and tested by an enzyme cutting technique in vitro. The pIRES2-EGFP transferred MSCs by means of electroporation. Transfer ction efficiency and transient expression were evaluated by fluorescent microscopy and flow cytometry. Results Flow cytometry analysis showed that (32.8%±3%) of the cells were positive for EGFP at 48 h after electroporation. The growth curves of passage 3 MSCs and that after electroporation were similar, uniformly expressing CD29, CD44, CD106, and CD54. Conclusion The efficiency of transfecting pIRES2-EGFP into MSCs could be considerably increased with optimized electroporation and electroporation procedure did not change the biological behavior of rMSCs.

关 键 词：骨髓基质干细胞 细胞培养 绿色荧光蛋白 基因转染 电穿孔 

分 类 号：R322.812[医药卫生—人体解剖和组织胚胎学]