大鼠骨髓间充质干细胞向软骨、脂肪和神经元样细胞分化的研究

目的：探讨体外大鼠骨髓间充质干细胞（rMSCs）多向分化潜能。方法：从大鼠骨髓中获得间充质干细胞，体外培养传代。通过地塞米松、TGF-β1和维生素C诱导rMSCs向软骨细胞分化，甲苯胺蓝染色和免疫细胞化学检测Ⅱ型胶原进行鉴定。通过地塞米松和胰岛素诱导rMSCs向脂肪细胞分化，采用苏丹Ⅳ染色鉴定。在含IBMX、GDNF和IL-1β的培养基中诱导rMSCs向神经元样细胞分化，免疫细胞化学方法检测神经细胞特异标志NSE和MAP-2a,b。结果：rMSCs被诱导7 d后，分化成软骨细胞，甲苯胺蓝异染阳性、Ⅱ型胶原阳性。被诱导10 d后，分化成脂肪细胞，苏丹Ⅳ染色阳性。被诱导7 d和15 d后，分化成神经元样细胞，表达NSE和MAP-2a,b，15 d后NSE阳性率是(20.060±0.790)%，MAP-2a,b阳性率是(17.364±5.738)%。结论：体外rMSCs被诱导分化成软骨细胞、脂肪细胞和神经元样细胞，证明其具有多向分化潜能, 是组织工程研究中最有前途的种子细胞之一。

Study on differentiation of rat bone marrow mesenchymal stem cells into chondrocytes, adipocytes, and neuron-like cells

Objective To explore the multipotency of rat bone marrow mesenchymal stem cells (rMSCs) in vitro. Methods rMSCs were obtained from rat bone marrow, cultured and passaged. rMSCs were induced to differentiate into chondrocytes in induction medium containing desamethasone, TGF-β1 and vitamin C. Then differentiated cells were identificated by toluidine blue stain and immunocytochemistry for collagen type Ⅱ. rMSCs were induced to differentiate into adipocytes in induction medium containing desamethasone and insulin. Then differentiated cells were identificated by Sudan IV stain. rMSCs were induced to differentiate into neuron-like cells in induction medium containing IBMX,GDNF, and IL-1β. Then neuron specific markers, such as NSE and MAP-2a,b, of the differentiated cells were identified by immunocytochemistry. Results rMSCs differentiated into chondrocytes on the 7th day after being induced. Toluidine blue metachromatic staining of differentiated chondrocytes in induction media was positive. Additionally, many differentiated chondrocytes in the experimental groups expressed collagen type Ⅱ. In vitro rMSCs differentiated into adipocytes and lipid droplets were scarlet red by Sudan Ⅳ staining on the 10th day after being induced. rMSCs differentiated into neuron-like cells and expressed NSE and MAP-2a,b by immunocytochemistry on the 7th day and the 15th day after being induced. The percentage of positive cells of NSE was (20.060±0.790)% and the percentage of positive cells of MAP-2a,b was (17.364±5.738)% on the 15th day.Conclusion rMSCs can differentiate into chondrocytes, adipocytes, and neuron-like cells in vitro, which shows that rMSCs have multipotency and are one of the best seeds cells in the tissue engineering.