程序性坏死阻断剂对铝致神经细胞凋亡的影响及其机制  

Effect of necrostatin-1 on apoptosis induced by aluminum and its mechanism

作　　者：张勤丽[1] 吉秀亮[1] 郭卫力[1] 张策[1] 刘承芸[1] 牛侨[1] 

机构地区：[1]山西医科大学公共卫生学院,太原030001

出　　处：《中华劳动卫生职业病杂志》2010年第3期175-180,共6页Chinese Journal of Industrial Hygiene and Occupational Diseases

基　　金：国家自然科学基金资助项目（30371203,30671777,30740032）

摘　　要：目的 了解程序性坏死阻断剂necrostatin（Nee-1）对铝诱导的神经细胞凋亡的影响,并探讨Nec-1对Caspases凋亡通路的作用机制.方法 用4 mmol/L铝染毒神经母细胞瘤细胞株（SH-SYSY）制备铝致神经细胞死亡模型,在不同浓度铝和（或）Nec-1作用下检测细胞活力的变化,用Heochst 33342/碘丙啶（PI）双染法观察细胞的凋亡和坏死现象,Annexin V/PI双染法定量细胞的凋亡率和坏死率.用Caspase-3、Caspase-8、Caspase-9酶活力的变化检测凋亡通路,用免疫细胞化学法检测核转录因子（NF-κB）和细胞色素c（Cyt-c）蛋白表达的变化.结果 随着铝染毒剂量的加大,细胞活力明显下降;但加入60 μmol/L Nec-1后,细胞活力明显增强.差异有统计学意义（P〈0.05）. Nec-1浓度的提高使细胞活力明显上升,表现了明显的促进神经细胞生长、抑制细胞死亡的作用.凋亡和坏死的荧光显微镜观察及流式细胞术定量检测结果 表明,随着铝染毒剂量的增加,神经细胞的凋亡率与坏死率明显上升,但在0～90μmol/L Nec-1作用下,Nec-1浓度越高,细胞的坏死率降低越明显,差异均有统计学意义（P〈0.05,P〈0.01）.同时,虽然Nec-1是程序性坏死的阻断剂,但其对凋亡率也有明显的影响,可以使染铝细胞的凋亡率明显下降,差异有统计学意义（P〈0.05,P〈0.01）.对Nec-1作用后细胞的凋亡相关酶Caspase-3、Caspase-8、Caspase-9活力变化检测结果 表明,Nec-1对Caspase-9活力的影响不明显,且对线粒体通路的凋亡诱导分子Cry-c的作用也不明显;但对Caspase-8活力的影响明显,表现在可以明显降低各染铝细胞的Caspase-8活力,提高NF-κB蛋白表达量和在高剂量染铝细胞中降低Caspase-3的活力.结论 Nec-1可以降低铝诱导的神经细胞凋亡,并通过死亡受体通路Caspase-8上调NF-κB表达,降低细胞凋亡率.Objective To study the effect of necroslatin （Nee-1） on apoptosis induced by aluminum （A1）, and approach the mechanism. Methods Neural cell death model was made by 4 mmol/L Al treated neuroblastoma cells （SH-SY5Y）. Cell viabilities were detected at different concentrations of Al and/or Nec-1. Heochst 33342 / PI double staining was used to observe apoptosis and （or） necrosis that were quantified by flow cytometry using Annexin V / PI double staining. Apoptotic pathway was tested by activities of Caspase-3, Caspase-8 and Caspase-9. In addition, the expression of NF-kB and Cyt-c was measured by immunocytochem-istry.Results Cell viabilities were significantly decreased with the increasing concentrations of Al （P〈0.05）, which could be significantly upregulated by 60 μmol/L Nec-1 （P〈0.05） and were correlated with the concentrations of Nec-1 （P〈0.05, P〈0.01）. Apoptosis and necrosis were observed under fluorescent microscope and quantified by flow cytometry, which suggested an increasing trend of apoptotic and necrotic rates （P〈0.05, P〈 0.01）. Whereas, Nec-1 could not only decrease the necrotic rate but also apoptotic rate as well （P〈0.05, P〈 0.01）. Data of Nec-1 on caspases activities showed that Nec-1 could not affect Caspase-9 activity （P〉0.05） and Cty-c protein expression as well （P〉0.05）. However, Nec-1 could reduce Caspase-8 activity significantly （P〈 0.05, P〈0.01） and increase NF- k B protein expression（P〈0.05, P〈0.01） and finally decrease Caspase-3 activity （P〈0.05 ）. Conclusion Nec-1 could reduce cell apoptosis induced by Al, through Caspase-8 pathway, and up-regulate the expression of NF- k B protein.

关 键 词：铝 神经元 脱噬作用 CASPASE类 

分 类 号：R686[医药卫生—骨科学]