【摘要】 目的探讨人脐带间充质干细胞条件培养液(human umbilical cord mesenchymal stem cells conditioned medium,hUCMSCs-CM)对β样淀粉蛋白(amyloidβ,Aβ)损伤原代前额叶皮质神经元凋亡的影响及线粒体凋亡途径通路在其中的作用。方法进行大鼠前额叶皮质神经元原代培养,第7～8天的神经元用于实验。分离并传代培养人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,hUCMSCs),培养3～4代的细胞用于收集条件培养液(conditioned medium,CM);通过MTT实验检测神经元活力;通过Hoechst荧光染色观察各组神经元凋亡情况;通过Western blot检测神经元cyt-c及cleaved caspase-3蛋白表达水平。结果 Aβ作用于体外培养7 d的皮质神经元12 h后,5μmol/L Aβ、10μmol/L Aβ、20μmol/L Aβ组相对细胞活力显著低于溶剂对照组(P<0.05)。10μmol/L Aβ作用于培养7 d的皮质神经元后,6,12,24 h组相对细胞活力显著降于溶剂对照组(P<0.05)。hUCMSCs-CM作用于Aβ损伤前额叶皮质神经元12 h后,中、高剂量组相对细胞活力高于Aβ损伤组(P<0.05)。Aβ损伤组皮质神经元凋亡率高于溶剂对照组,hUCMSCs-CM低、中、高剂量组皮质神经元凋亡率低于Aβ损伤组,差异有统计学意义(P<0.05)。Aβ损伤组前额叶皮质神经元cyt-c及cleaved caspase-3蛋白表达水平显著高于溶剂对照组;hUCMSCs-CM低、中、高剂量组前额叶皮质神经元cyt-c蛋白表达水平低于Aβ损伤组,hUCMSCs-CM中、高剂量组前额叶皮质神经元cleaved caspase-3蛋白表达水平低于Aβ损伤组,差异有统计学意义(P<0.05)。结论 hUCMSCs-CM通过抑制Aβ引起的cyt-c、cleaved caspase-3蛋白表达增加对抗Aβ诱导的原代培养皮质神经元凋亡,进而发挥神经保护作用,其神经保护作用与线粒体凋亡通路有关。更多还原

【Abstract】 Objective To investigate the protective effect of human umbilical cord mesenchymal stem cells conditioned medium(hUCMSCs-CM) against amyloid β(Aβ) induced apoptosis in cultured rat prefrontal cortical neuron and the mechanism: down-regulation of the expression of cyt-c and cleaved caspase-3 thus inhibiting the mitochondrial apoptotic pathway. Methods Human umbilical cord mesenchymal stem cells P3 and P4 were used to collect the hUCMSCs-CM. Prefrontal cortical neurons of rats cultured 7d were treated with Aβ and/or hUCMSCs-CM, respectively. The neuron viability was determined by MTT assay, and the neuron apoptosis was tested by Hoechst staining. The expression of cyt-c and cleaved caspase-3 were investigated by Western blot. Results Compared with vehicle-control group, Aβ(5, 10, or 20 μmol/L Aβ treated 12 h; or 10 μmol/L Aβ treated 6 h, 12 h, or 24 h) decreased the neuron viability(P<0.05)and increased the neuron apoptosis(P<0.05). Compared with Aβ group, hUCMSCs-CM increased the neuron viability(10% and 20%, treated 12 h; P<0.05)and decreased the neuron apoptosis(5%, 10% and 20%, treated 12 h; P<0.05). Compared with vehicle-control group, Aβ up-regulated the expression of cyt-c(P<0.05) and cleaved caspase-3(P<0.05). Compared with Aβ group, hUCMSCs-CM treatment alleviated the up-regulation of cyt-c(5%, 10% and 20% hUCMSCs-CM treated 12 h; P<0.05) and cleaved caspase-3(10% and 20% hUCMSCs-CM treated 12 h; P<0.05) in a dose-dependent manner. Conclusion hUCMSCs-CM protected against Amyloid β induced apoptosis in cultured rat cortical neuron partly through mitochondrial apoptotic pathway.更多还原