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目的探讨由丹参诱导骨髓间充质干细胞(mesenchymal stem cells,MSCs)定向分化的神经细胞是否具有突触循环功能。方法采用优化诱导方案对4～5代状态好的MSCs进行反复多次诱导,终止诱导后分别进行免疫荧光细胞化学、细胞内钙离子流及突触功能检测。以50 mmol/L KCl作为诱发动作电位的刺激因子,采用激光共聚焦显微镜(LSCM)对Fluo-3/AM和SynaptoRed C-2荧光探针进行荧光激发,检测分化神经细胞在细胞外高钾刺激下细胞内钙流及细胞突触功能。结果4次诱导5 h后,神经样细胞伸出突起并交互成复杂网状;免疫荧光细胞化学显示4次诱导5 h后TUJ-1表达率为(96.7±2.8)%,突触小泡蛋白(synaptophysin)表达率为(96.2±2.1)%;LSCM显示4次诱导5 h后细胞在高钾刺激下细胞内钙流增加,细胞突触发生胞吞胞吐。结论丹参可高效快速诱导MSCs分化为神经细胞,此细胞具有正常的突触循环功能。
Objective To investigate whether the neural cells differentiated from mesenchymal stem cells (MSCs) induced by Salvia miltiorrhiza have synaptic circulation function. METHODS: The optimized induction protocol was used to repeatedly induce MSCs in the 4th to 5th generation. After induction, immunofluorescence cytochemistry, intracellular calcium currents, and synaptic function were detected. 50 mmol/L KCl was used as the evoked action potential stimulating factor. Fluorescence excitation of Fluo-3/AM and SynaptoRed C-2 fluorescent probes was performed by confocal laser scanning microscopy (LSCM) to detect extracellular potassium stimulation in differentiated neurons. Under the intracellular calcium flow and cell synapse function. Results After 5 h induction, the neuron-like cells protruded and interacted into a complex network. Immunofluorescence cytochemistry showed that after 5 h induction, the expression rate of TUJ-1 was (96.7±2.8)%. Synaptophysin The expression rate of (synaptophysin) was (96.2±2.1)%; LSCM showed that after 4 times of induction, the intracellular calcium flux was increased in cells stimulated with high potassium, and synaptic cell exocytosis and exocytosis occurred. Conclusion Salvia miltiorrhiza can effectively induce MSCs to differentiate into nerve cells. This cell has normal synaptic circulation function.