Bcl-2蛋白在蛋白磷酸酶抑制剂冈田酸诱导的体外神经细胞退化中的作用  

EFFECT OF BCL-2 OVEREXPRESSION ON OKADAIC ACID-INDUCED NERVE CELL APOPTOSIS IN VITRO

作　　者：陈丽琴[1] 

机构地区：[1]内蒙古医学院基础医学院,内蒙古呼和浩特010059

出　　处：《内蒙古医学院学报》2008年第1期6-12,共7页Acta Academiae Medicinae Neimongol

基　　金：上海市研究和发展基金(04DZ00415)

摘　　要：目的:在体外水平条件下,探讨抗凋亡蛋白Bcl-2在蛋白磷酸酶抑制剂冈田酸所诱导的阿尔茨海默病(Alzheimer disease,AD)样神经细胞退化中是否具有细胞保护的作用。方法:以人神经母细胞瘤细胞SH-SY5Y作为体外神经细胞研究材料,采用脂质体介导重组质粒pEGFP-N1-Bcl-2瞬时转染细胞的方法,造成神经细胞外源性过表达Bcl-2蛋白与绿色荧光蛋白的融合蛋白,以荧光显微镜下观察到绿色荧光蛋白显示转染阳性细胞,再用冈田酸处理转染细胞,通过DNA荧光染料染色法显示细胞凋亡核形,显微镜下计数细胞测定凋亡率。结果:①冈田酸剂量依赖性地引起SH-SY5Y细胞凋亡,DNA荧光染料染色显示,80nmol冈田酸作用24h引起该种细胞出现多量核浓聚、核碎裂的凋亡核形;②脂质体介导pEGFP-N1-Bcl-2和空质粒pEGFP-N1瞬时转染SH-SY5Y细胞,细胞分别过表达Bcl-2-绿色荧光蛋白的融合蛋白和绿色荧光蛋白,前者主要分布于近核的胞质,而后者则在胞质和胞核中均有分布;200nmol经典促凋亡剂星形孢菌素处理细胞24h,Bcl-2重组质粒转染组的细胞凋亡率显著低于空质粒转染组的细胞凋亡率(P<0.01);③80nmol冈田酸处理细胞24h,Bcl-2重组质粒转染组的细胞凋亡率显著低于空质粒转染组的细胞凋亡率(P<0.05)。结论:冈田酸能引起体外培养神经细胞SH-SY5Y发生凋亡性退化,Bcl-2重组质粒瞬时转染引起该种细胞过表达具有抗凋亡活性的融合蛋白Bcl-2-绿色荧光蛋白,Bcl-2过表达能部分抑制冈田酸所致SH-SY5Y细胞退化,这提示Bcl-2在冈田酸所致阿尔茨海默病样神经细胞退化过程中可能具有细胞保护作用。Objective：To explore the effect of anti - apoptotic Bcl - 2 protein on Okadaic acid （OA） -induced nerve cell apoptosis in vitro. Methods：Human SH -SY5Y neuroblastoma cells were applied to investigate the influence of exogenous Bcl -2 overexpression on apoptotic nerve cell induced by OA. MTF reduction assay was used to determine cell viability after OA treatment. Lipofectamine was used to mediate tansfection of recombinant plasmid pEGFP - N1 - Bcl - 2 into SH - SY5Y cells. Exogenous Bcl- 2 -GFP （green fluorescent protein, GFP） fusion protein was observed by fluorescent microscopy. Apoptotic nerve cell was detected by DNA fluorescent dye Hoechst 33258 staining. Re-sults：1. OA decreased cell viability of SH- SY5Y cells in dose dependent manner. Hoechst staining showed that a lot of chromatin condensation and fragmentation typical for apoptosis appeared following 24 h of 80 nmol OA treatment. 2. Exogenous Bcl -2 -GFP induced by recombinant plasmid transfection mainly resided in peri -nucleus cell bodies, while exogenous GFP induced by vector transfection located in cytosol, nuclei and processes. Hoechst staining showed that apoptotic Bcl -2 transfected cells were markedly less than apoptotic vector tmnsfected ceils after 24 h of 200 nmol staurosporine ex- posure（P 〈0. 01 ）. 3. Hoechst staining further revealed that apoptotic Bcl -2 transfected ceils were markedly less than apoptotic vector transfected cells after 24 h of 80 nmol OA exposure （ P 〈 0. 05）. Conclusion：Exogenous Bcl- 2 overexpression partially inhibited OA -induced apoptosis in SH - SY5Y nerve ceils, suggesting that Bcl -2 related apoptotic regulatory mechanism is possibly involved in OA -induced neurodegenerative pathway and that Bcl- 2 had nerve protective role in it.

关 键 词：BCL-2蛋白 神经细胞 凋亡 冈田酸 转染 

分 类 号：R31[医药卫生—基础医学]