绿色荧光蛋白标记大鼠间充质干细胞的实验研究

目的研究重组腺病毒载体介导绿色荧光蛋白基因转染大鼠间充质干细胞（MSCs）的方法。方法 用密度梯度离心法分离纯化大鼠MSCs并进行鉴定，以携带绿色荧光蛋白基因的重组腺病毒载体Ad-GFP转染MSCs，流式细胞仪检测即时、15 d和30 d的标记率；通过流式细胞仪检测细胞周期、凋亡率，CCK-8法检测增殖能力，明确标记后细胞的生长特性；通过流式细胞仪检测标记后细胞表面标志（CD29、CD34、CD44和CD45）表达的变化。结果 Ad-GFP对大鼠MSCs的即时标记率高达89.71%，15 d和30 d的标记率分别为：85.10%、82.92%。标记后细胞周期的分布、凋亡率、增殖能力与未标记细胞相比，差异无显著性（P>0.05）。标记后细胞表面标志的表达亦不受影响。结论 重组腺病毒载体Ad-GFP能高效标记MSCs，且标记后细胞的增殖能力不受影响，表面标志的表达不发生改变。

A study on rat bone marrow mesenchymal stem cells labeling with green fluorescent protein

Objective To investigate an efficient method to transfect green fluorescent protein gene（GFP） mediated by recombinant adenovirus vector（Ad-GFP） to rat bone marrow mesenchymal stem cells（MSCs） in vitro. Methods Wistar rats’ bone marrow derived MSCs were separated and purified in vitro by Percoll density gradient centrifugation method and identified by flow cytometry（FCM）. Labeled MScs were observed and the immediate, 15-day and 30-day labeling efficiency were assessed by flow cytometry（FCM）. Cell cycle and apoptosis ratio of labeled MSCs were revealed, the growth curves were delineated, so as to identify whether the marker cast an influence on the cell growth characteristics in vitro. Furthermore, the surface markers(CD29、CD34、CD44、CD45) of labeled MSCs were detected through flow cytometry and were compared with those of non-labeled MSCs. Results the immediate, 15-day and 30-day labeling efficiency were 89.71%, 85.10% and 82.92% respectively. There was no statistically difference in cell cycle, apoptotic ratio and proliferative ability between the labeled MSCs and non-labeled MSCs（P>0.05）. The marker didn’t influence the expressions of surface markers(CD29、CD34、CD44、CD45) of rat MSCs. Conclusions Transgection with Ad-GFP is a highly effective method for labeling MSCs and have no effect on the growth characteristics and expressions of surface markers of rat MSCs cultured in vitro.