丹酚酸B对人脐带间充质干细胞向胆碱能神经元分化的影响

Effect of Salvianolate on differentiation of human umbilical cord mesenchymal stem cells into cholinergic neurons

目的:评估不同剂量丹酚酸B联合脑源性神经营养因子(BDNF)对人脐带间充质干细胞(hUCMSCs)向胆碱能神经元分化的影响。方法:流式细胞技术鉴定培养的hUCMSCs细胞表型;MTT法评估丹酚酸B浓度对hUCMSCs细胞增殖生存率的影响;观察3种不同剂量丹酚酸B联合BDNF诱导hUCMSCs分化后的细胞形态学变化;ELASA法测定培养上清液中乙酰胆碱(Ach)浓度,免疫组化法测定细胞神经巢蛋白(Nsetin)、神经元特异性烯醇化酶(NSE)和胆碱乙烯转移酶(ChAT)表达水平。结果:hUCMSCs细胞表型具有干细胞的表型特征,丹酚酸B随着作用浓度的增大对hUCMSCs的增殖具有抑制作用。丹酚酸B联合BDNF诱导分化后的hUCMSCs可见神经元样分化及轴突形成的网状结构;上清液Ach浓度和细胞Nestin、NSE及ChAT表达率,在中、高剂量联合组中的表达均高于低剂量联合组和BDNF组(P<0.05),而中、高剂量联合组之间差异不显著。结论:丹酚酸B联合BDNF能提高hUCMSCs向胆碱能神经元分化效率,丹酚酸B的最宜诱导分化浓度为50 mg/L。

Objective： To investigate the effect of salvianolate combined with brain-derived neurotrophic factor （BDNF） on the differentiation of human umbilical cord mesenchymal stem cells （hUCMSCs） into cholinergic neurons. Methods： The cell phenotype was detected by flow cytometry. The effect of salvianolate on survival rate of hUCMSCs was detected by MTr assay. We observed the morphologic changes of the cells induced by three concentrations salvianolate combined with BDNF. Further, we examined the contents of Ach in the culture supernatants with ELASA and the positive expression rates of Nestin, neuron-specific enolase （NES） and choline acetytransferase （CHAT） in the differentiated cells. Results： Flow cytometry analysis showed that hUCMSCs had the characteristic of stem cell. Salvianolate inhibited the proliferation of hUCMSCs under high concentrations. Ceils showed a neuren-like morphology when treated with BDNF or salvianolate combined BDNF. The contents of Ach and positive expression rates of Nestin, NES and ChAT in high-dose and middledose combination groups were significantly higher than those in low-dose combination group and BNDF group （P 〈 0.05 ）. There was no significant difference between the high-dose and middle-dose combination groups （ P 〉 0.05 ）. Conclusion：Salvianolate combined with BDNF would enhance hUCMSCs differentiating into cholinergic neurons, and the optimal concentration of salvianolate was 50 mg/L.