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骨髓基质细胞的分离培养及转基因标记

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Keywords: 骨髓基质细胞,增强型绿色荧光蛋白,流式细胞术

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Abstract:

目的采用密度梯度离心法进行骨髓基质细胞(MSCs)的分离培养,结合体外转入增强型绿色荧光蛋白(EGFP)基因标记方法,可提供大量纯度高、便于观察的种子细胞。方法取成年SD大鼠四肢骨及胸骨获取骨髓细胞冲洗液,密度梯度离心法分离后培养,HE染色观察细胞核浆比,流式细胞术测定细胞纯度;用逆转录病毒载体转染EGFP标记基因,G418筛选后传代培养,流式细胞术检测EGFP的表达率。结果原代培养的MSCs形态多样,增殖迅速,随着培养时间的延长,细胞增殖变慢,形态趋于同一,以梭形细胞为主,核浆比高;流式细胞术检测发现CD90+/CD45-/CD11b-细胞达到75%,细胞在体外能够稳定地表达EGFP,表达率达到55.16%。结论运用密度梯度离心法分离培养的MSCs纯度高,增殖能力强;体外转入EGFP基因是一种方便、特异性高的标记MSCs的方法,为脑损伤修复研究提供了依据。

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