慢病毒介导的中性内肽酶对Aβ诱导的SK-N-SH细胞凋亡的影响

Effects of neprilysin mediated by lentiviral vector on amyloid beta peptide-induced SK-N-SH cell apoptosis

目的:探讨脑中性内肽酶(NEP)外源表达对神经毒物质β淀粉样肽(Aβ)诱导SK-N-SH细胞凋亡的影响。方法:采用脂质体法将含中性内肽酶NEP的四质粒系统慢病毒载体转染293FT包装细胞,制备高滴度病毒载体,感染Aβ处理的SK-N-SH细胞,用Western blotting方法检测NEP对Aβ的降解作用、MTT法检测细胞存活率、流式细胞术分析细胞凋亡情况、RT-PCR技术测定凋亡相关基因bcl-2及bax的表达及caspase-3活性检测。结果:胞内高表达的NEP能够显著降解Aβ,其降解程度与NEP的表达量有剂量依赖关系。细胞存活率及流式细胞术检测结果显示NEP高表达可减轻Aβ诱导的细胞凋亡,细胞存活率于感染病毒后48 h最强,为对照的170%(P<0.01),NEP活性组的凋亡率为3.86%,低于对照组的6.41%;RT-PCR结果显示NEP对Aβ诱导的细胞凋亡保护作用可能是通过减少bax的表达,抑制了caspase-3的激活程度来实现的。结论:NEP可以降解Aβ,可以保护由Aβ沉积所致的神经细胞凋亡。

AIM ： To explore the protective effects of neprilysin on SK - N - SH cell apoptosis induced by amyloid beta - peptide （Aβ）. METHODS ： The four - plasmid lentiviral vector including packaging plasmids were cotransfected into human embryonic kidney 293FT cells by using LipofectaminTM 2000. The packaged virus was harvested 72 h later. Then SK - N - SH cells pretreated with Aβ were transfected with the obtained virus. Apoptotic cells were measured by MTT assay, caspase - 3 assay and flow cytometric analysis. Apoptosis - associated gene bax and bcl - 2 in cells were detec- ted by reverse transcription PCR （ RT - PCR）. Western blotting was used to detect expression of neprilysin and Aβ levels. RESULTS ： Ectopic expression of NEP enhanced neurocyte cell growth though degrading Aβ. The level of Aβ decreased in a dose dependent manner with NEP. MTT and flow cytometry indicated that expression of NEP reduced the cell apoptosis induced by Aβ The cell survival rate was 170% higher than that in control after 24 h lentivirus infection. Apoptotic rate in NEP group was 3. 86% and in control group was 6.41%. RT- PCR showed that protective effect of NEP on the Aβ -induced apoptosis achieved by reducing the expression level of bax, and inhibiting caspase - 3 activity. CONCLUSION ： Ectopic expression of NEP protects neurons against Aβ - induced apoptosis.