大鼠骨髓间充质干细胞的分离培养及GFP转染标记

目的建立大鼠骨髓间充质干细胞(MSCs)的分离和培养方法,检测绿色荧光蛋白基因转染MSCs的瞬时表达及转染效率。方法应用Percoll密度梯度离心法分离大鼠MSCs,贴壁法不断纯化,流式细胞仪检测2代细胞表面CD34、CD71和CD90的表达,然后用pEGFP-N3与Lipofectamine 2000不同浓度比例转染MSCs,荧光倒置显微镜下观察瞬时表达及转染效率。结果①原代MSCs多呈纺锤形或梭形,有聚集生长的倾向,多3~5个细胞成为一个集落。传代后,细胞变为形态均一的排列有序的成纤维细胞样,长梭形,胞浆突起较少,胞体轮廓不甚清晰,胞体也相对较大;②GFP转染后24 h即可见绿色荧光蛋白表达,72 h表达最强,此后逐渐减弱,到4周时仍可见少量表达;③转染效率与质粒和脂质体的浓度比例有关,1∶2到1∶3最高。结论利用Percoll密度梯度离心法结合贴壁法可以获得比较纯的MSCs;GFP转染是一种较好的MSCs标记方法。

Rat mesenchymal stem cell's culture and label with green fluorescent protein gene transfection

Objective To establish a method of isolation and culture of the rat mesenchymal stem cells(MSCs),and to investigate the transient expression of green fluorescent protein gene transfection into MSCs.Methods MSCs,which were initially isolated from the bone marrow of rats,were cultured in vitro and tested by flow cytomertry.Then they were transfected with pEGFP-N_(3) by means of Lipofectamine 2000.The ratio of plasmid to Lipofectamine varied according to the experiment design.The results of the transient expression of GFP and transfection efficiency were observed by fluorescence microscope.Results MSCs were adherent,elongated,and spindle-shaped in the primary culture after 24 hours of plating,and then became several little clones;During mitosis the cells regained a rounded appearance,and remained loosely attached until division was completed.When they reached 80% confluence,MSCs were subcultured and gained uniform shape and similar growth pattern.The expression of GFP began at 24 hours after transfection,and reached the peak at 72 hours and decreased in 1 week;however,the transient expression remained for more than 4 weeks.Transfection efficiency was correlated with the ratio of plasmid to lipofectamine.Conclusion High pured MSCs can be obtained by Percoll gradient centrifuging and adherent method.GFP transfection is a better method to label MSCs.