神经干细胞过表达Hsp75降低Aβ介导的神经毒性

摘要

AIM:To investigate the effect of heat shock protein 75 ( Hsp75 ) over-expression on Aβ-induced neurotoxicity in the neural stem cells and to explore its mechanism.METHODS:An adenovirus-mediated Hsp75 over-ex-pression vector was used in vitro.The mouse neural stem cell C17.2 was cultured in vitro and divided into control group, Aβgroup, negative adenovirus vector transfection group and Hsp75 over-expression adenovirus vector transfection group. The transfection and cellular immune identification were detected by fluorescence microscopy.The cell morphology was ob-served under inverted phase-contrast microscope.The cell viability and apoptosis were detected by MTT assay and flow cy-tometry, respectively.Hsp75 over-expression and cleaved caspase-3 protein level were measured by Western blot.RE-SULTS:Observation by fluorescence microscopy indicated that C17.2 cells were successfully transfected and Hsp75 gene was effectively expressed in the neural stem cells after transfection.In addition, the morphology and viability of the cells did not change and these cells did not differentiate after transfection.As compared with control group, the cell viability in Aβgroup and negative adenovirus vector transfection group was significantly decreased (P<0.05), and the cell apoptotic rate and cleaved caspase-3 level (P<0.05) were increased.As compared with Aβgroup and negative adenovirus vector transfection group, Hsp75 over-expression significantly increased the cell viability, and decreased the cell apoptosis and cleaved caspase-3 level ( P<0.05 ) .CONCLUSION: Hsp75 over-expression protects the neural stem cells against Aβ-induced injury.The mechanism may be related to inhibiting caspase-3 pathway-dependent apoptosis.%目的：应用腺病毒为载体在体外过表达热休克蛋白75（Hsp75），研究Hsp75蛋白过表达对神经干细胞在Aβ诱导神经毒性中的作用，并初步探讨其作用机制。方法：体外培养小鼠神经干细胞C17.2，实验分为对照组、Aβ处理组、腺病毒阴性感染组和腺病毒Hsp75过表达感染组。使用荧光显微镜观察腺病毒的感染情况并进行细胞免疫鉴定；倒置相差显微镜观察各组神经干细胞的形态；MTT法检测细胞活力；流式细胞术检测细胞凋亡率；Western blot检测Hsp75和活化型caspase-3蛋白水平。结果：荧光显微镜观察和Western blot检测表明腺病毒成功感染神经干细胞并高效表达Hsp75蛋白。此外，腺病毒感染不会导致细胞形态改变及细胞分化，同时也不会影响细胞活力。与对照组比较，Aβ处理组及腺病毒阴性感染组细胞存活率显著降低（P＜0.05），细胞凋亡率及活化型caspase-3蛋白水平显著增高（ P＜0.05）；然而，Hsp75过表达能显著提高神经干细胞的存活率，减少细胞凋亡和降低活化型caspase-3蛋白水平（P＜0.05）。结论：Hsp75过表达对Aβ诱导损伤的C17.2细胞具有明显保护作用，其机制可能是与抑制caspase-3途径依赖的细胞凋亡有关。