小鼠骨髓间充质干细胞的分离培养及其诱导分化为肺泡上皮细胞

Isolation and culture of mouse bone marrow mesenchymal stem cells and induced differentiation into alveolar epithelial cells

目的小鼠骨髓间充质干细胞(BMSCs)的分离培养技术门槛高且重复成功率低,一定程度上制约了相关研究的顺利开展。文中尝试优化全骨髓贴壁法分离培养小鼠BMSCs并探索其诱导分化为肺泡上皮细胞的有效方法。方法采用全骨髓贴壁法培养C57BL/6小鼠胫、股骨骨髓内容物,根据克隆状细胞集落大小及数量,动态调整传代时机与比例进行传代培养。取第6代细胞,计数检测细胞增殖能力,流式细胞术检测相关表面标志物,使用小气道上皮细胞培养基(SAEpi CM)进行诱导分化。结果随着传代次数增加及BMSCs的纯度逐渐提高,第6代细胞增殖速度趋于稳定,流式细胞术分析显示,第6代细胞表达造血干细胞表面标志物CD117的表达率仅为0.008 2%,但高表达骨髓间充质干细胞表面标志物CD29和Sca-1(表达率分别为99.1%和88.5%)。经SAEpi CM培养的BMSCs呈上皮样改变,并表达肺泡细胞的特异表面标志物——肺表面活性物质相关蛋白C。结论根据克隆状细胞集落大小及数量调整传代时间与比例的全骨髓贴壁培养法可有效分离培养得到小鼠骨髓间充质干细胞,并具有分化为肺泡上皮细胞的潜能。

Objective It has traditionally been difficult to isolate and culture mouse bone marrow mesenchymal stem cells( BMSC),which has low success rate.And thus restricts the development of related research to some extent. We aimed to optimize the whole bone marrow adherent method for isolation and culture of mouse bone marrow mesenchymal stem cells and search for an effective method of inducing BMSCs to differentiate into alveolar epithelial cells. Methods Bone marrow contents harvested from the tibia and femur of C57 BL/6 mice were cultured based on the whole bone marrow adherent method. The timing and split ratios of passage were determined according to the size and number of cell colonies. After 6 passages,cells were counted to detect cell proliferation ability,surface markers were examined by flow cytometry and Small Airway Epithelial Cell Medium( SAEpi CM) was used to induce the differentiation of BMSCs. Results With the increase of passages and the purity of BMSCs,the proliferation of cells at passages 6 tended to be stable. Flow cytometry showed that they were strongly positive for bone marrow mesenchymal stem cell surface markers CD29 and Sca-1( 99.1%,88.5%),but almost negative for the surface marker of hematopoietic stem cells CD117( 0. 008 2%). BMSCs cultured in SAEpi CM showed an epithelium-like morphological change and expressed surfactant associated protein C,a specific marker of alveolar epithelial cells. Conclusion It is effective to isolate and culture mouse bone marrow mesenchymal stem cells by adjusting the timing and split ratios of passage according to the size and number of the clonal cell colonies,which possessed the potential to differentiate into alveolar epithelial cells.