微囊化软骨细胞诱导骨髓间充质干细胞的体外定向分化

摘要

BACKGROUND:Traditional coculture methods for directional differentiation of bone marrow mesenchymal stem cells, such as direct contact method and Transwel coculture system, appear to have low purity and slow proliferation. OBJECTIVE:To compare the inductive effect of microcapsule coculture system and traditional transwel coculture system on the differentiation of bone marrow mesenchymal stem cells. METHODS:The passage 2 microcapsuled chondrocytes and the passage 3 bone marrow mesenchymal stem cells harvested from rabbits were co-cultured at a ratio of 1:1 in a Transwel chamber. Another passage 2 chondrocytes and passage 3 bone marrow mesenchymal stem cells were co-cultured using traditional transwel coculture system. Pure bone marrow mesenchymal stem cells were served as controls. MTT assay was used to compare cellproliferation, toluidine blue staining and safranine O staining were used for observation of cartilage matrix synthesis, alcian blue staining and ELISA test were used to measure glycosaminoglycans and synthesis of type II col agen, respectively. RESULTS AND CONCLUSION:Compared with the traditional co-culture method, the microcapsule coculture system and pure culture method showed better cellproliferation (P<0.05). The levels of glycosaminoglycans and type II col agen were higher in the microcapsule coculture group than the traditional coculture group and pure culture group (P<0.05). Moreover, the microcapsule coculture group showed better outcomes in toluidine blue staining and safranine O staining than the traditional coculture group and pure culture group. These findings indicate that the microcapsule coculture system is more effective in the induction of bone marrow mesenchymal stem cells than traditional Transwel coculture system.%背景：以往诱导骨髓间充质干细胞定向分化的直接接触共培养法、Transwel 膜非接触共培养诱导法存在干细胞纯度低、增殖缓慢等不足。目的：对比分析微囊化共培养体系与传统Transwel 共培养体系对骨髓间充质干细胞的诱导效果。方法：实验组取第3代兔骨髓间充质干细胞与微囊化的第2代兔关节软骨细胞，按1∶1的比例在Transwel小室进行共培养，同时设置第3代兔骨髓间充质干细胞与第2代兔关节软骨细胞共培养于Transwel 小室的传统共培养组，以及纯干细胞培养组。MTT法对比3组干细胞的增殖率，甲苯胺蓝染色、番红花O染色观察软骨基质的合成，阿利新蓝染色法和Elisa法定量测量糖胺聚糖与Ⅱ型胶原蛋白的合成。结果与结论：传统共培养组细胞增殖率低于实验组与纯干细胞培养组(P<0.05)。实验组细胞糖胺聚糖和Ⅱ型胶原蛋白水平高于传统共培养组、纯干细胞培养组(P<0.05)，甲苯胺蓝染色、番红花O染色强度强于传统共培养组、纯干细胞培养组。表明微囊化的软骨细胞能够成功诱导骨髓间充质干细胞向成软骨方向定向分化，且诱导效果优于传统的Transwel 膜分离共培养法。