丹参诱导大鼠骨髓间充质干细胞分化为神经元样细胞中的钙离子检测

Detection of [Ca2＋]i in neuron-like cells from bone mesenchymal stem cell differentiation induced by salvia miltiorrhiza

目的检测大鼠骨髓间充质干细胞经丹参注射液诱导分化的神经元样细胞内钙离子浓度，以期为骨髓间充质干细胞应用于神经系统疾病的治疗提供理论依据。方法从成年大鼠骨髓中获取骨髓间充质干细胞，体外扩增培养．经碱性成纤维生长因子预诱导后施加10mL／L丹参注射液于骨髓间充质干细胞培养液中。运用免疫荧光检测神经元特异性核蛋白（NeuN）在诱导后细胞与经新生大鼠海马获取的体外培养海马神经元中的表达。激光共聚焦技术检测诱导后的细胞内钙离子浓度，并与原代培养海马神经元内的钙离子浓度进行比较。结果大鼠骨髓间充质干细胞经碱性成纤维生长因子和丹参注射液处理后．可表达NeuN，并具有神经元样的表型。诱导分化的神经元样细胞内钙离子浓度为984．75±79．51，原代培养海马神经元内钙离子浓度为769．42 ±60．93．两者比较差异无统计学意义（P〉0．05）。结论丹参注射液诱导骨髓间充质干细胞分化的神经元样细胞具有神经元的某些特征。

Objective To measure the concentration of intracellular free Ca2＋ （[Ca2＋]i） in neuron-like cells resulted from rat bone mesenchymal stem cell （BMSCs） differentiation induced by salvia miltiorrhiza injection and provide some theoretical basis for the BMSCs transplantation. Methods The rat BMSCs were separated from rat bone marrow and cultured in vitro. After induced by basic fibroblast growth factor and 10 mL/L salvia miltiorrhiza injection, the cells were identified with immunoiquorescence staining against NeuN. The same procedure was performed on primarily cultured hippocampal neurons. Then, the [Ca2＋]i of the differentiated neuron-like cells was determined and compared with primarily cultured hippocampal neurons. Results The BMSCs after induced by basic fibroblast growth factor and salvia miltiorrhiza injection expressed neuronal phenotypes similar to the cell appearance of neurons with NeuN. The average fluorescence intensity of the neuron-like cells derived from BMSCs was 984.75 ±79.51, while the average fluorescence intensity of the primarily cultured hippocampal neurons was 769.42±60.93. No significant difference was found between them （P〉0.05）. Conclusion The neuron-like cells from rat BMSCs differentiation induced by salvia miltiorrhiza injection possess certain neuronal properties.