大鼠骨髓间充质干细胞分离培养方法的比较

目的:探讨体外分离培养高纯度、高活力大鼠骨髓间充质干细胞(MMSC)的方法,为眼科组织工程提供种子细胞。方法:取清洁级SD大鼠,改良贴壁筛选法分离培养MMSC,采用MTT法检测MMSC活性,流式细胞术检测MMSC纯度。对比改良贴壁筛选法与密度梯度离心法,剪去干骺端法与骨干中间剪断法,不同月龄大鼠MMSC活性和纯度(CD44 CD34-,CD90 CD34-)的差别。结果:改良贴壁筛选法比密度梯度离心法获取的MMSC细胞数量多,细胞纯度差异无明显统计学意义。剪去干骺端法比骨干中间剪断法获取的MMSC细胞数量少,细胞纯度差异无明显统计学意义。在相同接种密度条件下,MMSC的增殖活力随大鼠年龄的增长而下降。结论:改良贴壁筛选法是一种简便易行的培养高活力、高纯度MMSC的方法。

Isolation and culture of rat marrow-derived mesenchymal stem cells

AIM:To explore a practical method of isolation of marrow-derived mesenchymal stem cells(MMSC) with high purity and powerful vitality from rats,and to offer plenty of seed cells for ophthalmologic tissue project.METHODS:MMSC number and vitality were detected by MTT cell proliferation assay and MMSC purity(CD44 CD34-,CD90 CD34-) was detected by flow cytometry.The differences of MMSC number and MMSC purity between modified adherence screening group and density gradient centrifugation group,metaphysis cutting group and diaphysis cutting group,and the difference of MMSC vitality among 1-month group,2-month group and 4-month group were investigated.RESULTS:MMSC number of modified adherence screening group was more than that of density gradient centrifugation group.The difference of MMSC purity between modified adherence screening group and density gradient centrifugation group was not statistically significant.MMSC number of metaphysis cutting group was less than that of diaphysis cutting group.The difference of MMSC purity between metaphysis cutting group and diaphysis cutting group was not statistically significant.The differences of MMSC vitality among 1-month group,2-month group and 4-month group was statistically significant at the same seed cell density.CONCLUSION:Modified adherence screening method is simple and practical to isolate and culture MMSC of high purity and powerful vitality in vitro.