摘要
目的:构建三维微环境复合体培养体系,探索骨髓源性间充质干细胞在支架内生长、分化的规律。方法:通过贴壁培养法建立稳定的骨髓源性间充质干细胞系,采用流式细胞仪检测细胞表面标志;将壳聚糖,β-甘油磷酸钠及羟乙基纤维素按比例混合制备温敏性水凝胶。选取第3代骨髓源性间充质干细胞接种支架培养2周后,加入心肌组织裂解液培养5d。倒置相差显微镜下连续观察细胞形态变化的特征,收集细胞支架复合物行苏木精—伊红染色,扫描电镜观察其超微结构。结果:骨髓源性间充质干细胞经流式细胞仪检测表达CD29,不表达CD34。温敏性水凝胶含交织成网格状的纤维样结构,网孔内含胶冻样物质。骨髓源性间充质干细胞接种后呈球形,折光性好,不仅分布于纤维样结构表面,还渗入网孔内生长;HE染色支架呈嗜酸性;扫描电镜下可见支架具有适宜的孔径,细胞在支架上分裂增殖活跃;添加心肌组织裂解液后,细胞逐渐拉长呈梭形。结论:在构建的三维微环境复合培养体系中,骨髓源性间充质干细胞与支架生物相容性良好;复合培养体系能较好的模拟体内微环境,促进骨髓源性间充质干细胞的增殖、分化。
Objective: To explore the biological characteristics of cell proliferation and differentiation of bone marrow-derived mesenchymal stem cells, the three-dimensional microenvironment composite culture model was constructed. Methods: We established BMSCs line through cell adherent method, and the surface anti-gens of the cells were analyzed by flow cytometry. Hermosensitive hydrogel β-glycerophosphate, hydroxyethyl cellulose were mixed at the particular ratio. BMSCs of the 3 passages were seeded in the scaf-folds and cultured for 2 weeks in vitro. And then,myocardium lysate was added to culture for 5 days. The change of cells? morphology was observed through inverted phase contrast microscope. The cell-scaffold was collected and performed with hematoxylin-eosin (HE) staining,and the ultrastructure of the cell-scaf-fold was observed by scanning electron microscope. Results : The expression of CD29 was positive and CD34 was negative in the BMSCs. The hydrogels showed fiber-like material interweaved three-dimensional net, and colloidal material filled with pores of the net. The BMSCs seeded in the scaffolds remained spheri-cal ? and BMSCs not only adhere on the surface of fiber-like material, but also grew in the pores of the net. HE staining showed that the scaffolds were eosinophilic. Through scanning electron microscope,we could observe that the scaffolds had suitable pore size, and the BMSCs had active proliferation when they grew in the scaffolds. After myocardium lysate was added, cells had changed from circular to fusiform cells. Con-clusion :BMSCs had highly biocompatibility with the scaffolds,the composite culture system could simu-late the tissue structure and promote the directed differentiation of the BMSCs in vitro.
出处
《广西医科大学学报》
CAS
2013年第4期511-514,共4页
Journal of Guangxi Medical University
基金
广西科技厅资助项目(No.桂科青0229009)
(No.桂科攻0592007-1G)
(No.桂科自0832157)
广西自然科学基金资助项目(No.2012GXNSFAA053126)
关键词
骨髓间充质干细胞
温敏性水凝胶
复合培养体系
bone marrow-derived mesenchymal stem cells ; hermosensitive hydrogel ; composite culture model