兔骨髓间充质干细胞的分离、培养与鉴定

背景：目前对骨髓间充质干细胞常用的分离方法有密度梯度离心法、贴壁筛选分离法。目的：联合应用密度梯度离心法和贴壁筛选分离法体外分离培养、扩增兔骨髓间充质干细胞，并对其进行鉴定。设计、时间及地点：对比观察的细胞学实验，于2007-10／2008-03在上海市第六人民医院中心实验室完成。材料：2月龄新西兰纯种大耳白兔6只用于骨髓间充质干细胞取材与原代培养，1．073kg／L的Percoll分离液。方法：实验采用Percol分离液利用密度梯度离心法及结合贴壁分离筛选法来分离、纯化骨髓间充质干细胞，在采用密度梯度离心法得到骨髓间充质干细胞后，经贴壁培养及反复换液纯化骨髓间充质干细胞。分别取第3，5，7，9代骨髓间充质干细胞，行细胞计数，绘制细胞生长曲线。主要观察指标：倒置显微镜下观察原代及传代细胞的形态、生长情况。采用CD44及CD34抗体进行间接免疫荧光标记鉴定培养的干细胞。CD44染色呈阳性，CD34染色呈阴性，说明所提取、纯化的细胞是骨髓间充质干细胞。结果：增殖传代的骨髓间充质干细胞呈长梭形均匀分布生长，形态比原代培养的细胞更均匀，细胞生长旺盛、增殖迅速，胞核明显，核仁清晰，核浆比例大，细胞形态均匀，平行排列呈螺旋状或漩涡状，传代至第5代时无明显变化。随传代次数的增加，细胞增殖能力逐渐下降，第3～5代细胞增殖能力强。所分离培养的细胞均表达CD44，不表达CD34。结论：在体外采用密度梯度离心及贴壁培养法可获得高纯度的兔骨髓间充质干细胞。

Isolation, culture and identification of rabbit bone marrow mesenchymal stem cells

BACKGROUND: Both density gradient centrifugation and adherence method arc frequently used to isolate bone marrow mesenchymal stem cells (BMSCs).OBJECTIVE: To investigate the approaches to isolate, culture and identify the rabbit BMSCs in vitro by the combination of den,ity gradient centrifugation and adherence method. DESIGN, TIME AND SETTING: Contrast cytological study, which was performed in Central Laboratory of Shanghai 6th People's Hospital between October 2007 and March 2008.MATERIALS: Six 2-week-old rabbits were selected for BMSCs preparation and primary culture; Percoll separating medium (1.073 kg/L) was also used for this study.METHODS: BMSCs were separated and purified with Percoll separating medium by density gradient centrifugation and adherence method. The three-, five-, seven-, and nine-passage BMSCs were counted for growth curve. MAIN OUTCOME MEASURES: Morphological features and growth states of primary and passage cells were observed under inverted microscope. Indirect immunofluorescence of CD44 and CD34 antibodies were used to examine the stem cells. CD44 staining was positive, and CD34 staining was negative, suggesting the extracting and purifying cells were BMSCs. RESULTS: The passage BMSCs were uniformly distributed like fusiform shape, which were more uniform than primary cultured cells. The BMSCs grew productively and proliferated rapidly; meanwhile, the nucleolus was clear, caryopla.sm was in a large proportion, morphological features were uniform, ceils like bostrychoid or whirlpool were arranged parallelly, and the five-pa.ssage cells were not changed remarkably. Proliferation was decreased gradually with the passage increasing; especially, the proliferation of three-five-passage cells was the strongest. The separated cells expressed CD44 but not CD34. CONCLUSION: High-purified rabbit BMSCs are obtained by both density gradient centrifugation and adherence method.