MSCs分离培养及定向分化为血管内皮细胞的研究

Experimental study of marrow mesenchymal stem cell isolation,culture and differentiation into vascular endothelial cell in vitro

目的建立分离和培养大鼠骨髓源间充质干细胞(MSCs)的方法及定向分化为血管内皮细胞诱导条件的优化。方法淋巴细胞分离液(比重1.077 g/ml)密度梯度离心法分离单个核细胞(MNC),在含10%胎牛血清L-DMEM培养基中培养,并传代扩增MSCs;选取状态较稳定的第4代细胞,分别用含10%胎牛血清和2%胎牛血清的诱导液(终浓度为10 ng/mlVEGF、2 ng/mlbFGF的DMEM培养液)继续培养,于第7、14天用流式细胞术分析CD34的表达情况,免疫组化法观察FVIII的表达情况。结果密度梯度离心结合贴壁培养法能有效分离纯化MSCs,细胞呈均一的成纤维细胞样,流式细胞术分析MSCs结果显示,CD34阳性率为1.01%、CD29阳性率为97.32%;诱导14 d后免疫组织化学法检测FVIII的表达,10%和2%胎牛血清诱导体系细胞的阳性率分别为12.21%和91.43%。结论密度梯度离心结合贴壁培养法能有效分离纯化MSCs;就诱导效果而言,2%胎牛血清诱导体系明显好于10%胎牛血清诱导体系,说明低浓度的胎牛血清更有利于MSCs向血管内皮细胞分化。

Objective To explore the rat marrow mesenchymal stem cells （MSCs） isolation, culture and discuss optimization of induction differentiation into vascular endothelial cell in vitro. Methods MSCs were isolated from healthy rat and purified by using density gradient centrifugation and adherent culture methods. Confluent cells （MSCs）from passages 4 （P4） were cultivated in six-pore plaque containing DMEM, 2% FCS or 10% FCS, 10 ng/ml VEGF, 2 ng/mlbFGF. Special antigen of FVIII and CD34 was tested after 7 days and 14days. Results MSCs can be isolated and purified by density gradient centrifugation and adherent culture methods, generally were spindle-shaped fibroblast-liked cells. FCM （flow cytometry） analysis showed that 97. 32% cells expressed CD29, 1.01% cells expressed CD34 which is a antigens of Hemopoietic stem cell. Immunohistochemical staining of FVⅢ showed that 12. 21% cells in 10% FCS expressed Endothehal Cells typical antigens FVIII after 14 days, but 91.43% cells in 2% FCS expressed FVⅢ. Conclusion It is a simple and practical method to separate MSCs from the bone marrow of adult rats by means of density gradient centrifugation and adherence to the culture plastic. MSCs differentiating into endothelial cells in 2% FCS were better than it in 10% FCS.