Tau蛋白去磷酸化对细胞凋亡的影响及其机制的研究
发布时间：2019-01-15 07:14
【摘要】： 目的:本课题组已证实tau蛋白过度磷酸化过程参与了神经元逃逸凋亡并发生退行性变性死亡,本研究将进一步研究tau蛋白去磷酸化过程在凋亡发生中的作用及可能机制。 方法:先将人最长的野生型tau(tau441)基因稳定转染到人肾母细胞瘤细胞(HEK293)内,以建立稳定过度表达tau的细胞株:HEK293/tau;再在HEK293细胞(HEK293/wt和HEK293/tau)中瞬时转染质粒PP2A使之过度表达PP2A,转染空载体pcDNA作为对照。接着分别用不同的凋亡诱导剂staurosporine,camptothecin,H2O2处理这两种细胞。在作用6小时后,用流式细胞计量术检测细胞凋亡率;用免疫印迹的技术检测细胞内PP2A、抗凋亡因子Bcl-2、tau蛋白的表达和活性水平,并分析Bcl-2、tau蛋白磷酸化变化的关系。 结果:1)过度表达PP2A使细胞可以抵抗凋亡诱导剂所诱导的细胞凋亡,同时细胞内抗凋亡因子Bcl-2的ser87位点磷酸化水平显著下降。2)在不同凋亡诱导剂的作用下:过度表达tau的HEK293/tau细胞凋亡率显著低于HEK293/wt细胞;当HEK293/tau同时过度表达PP2A时,凋亡率显著上升,同时tau蛋白在ser198/ser199/ser202位点发生去磷酸化,抗凋亡因子Bcl-2 ser87位点磷酸化水平显著升高;经相关性分析发现tau蛋白ser198/ser199/ser202位点的去磷酸化水平与抗凋亡因子Bcl-2的ser87位点磷酸化水平之间存在着正相关。 结论:过度表达PP2A可通过使抗凋亡因子Bcl-2在ser87位点去磷酸化而抵抗细胞凋亡。过度表达tau蛋白具有抵抗细胞凋亡的作用;而当tau蛋白被去磷酸化时,可能通过上调抗凋亡因子Bcl-2磷酸化水平而使细胞发生凋亡。
[Abstract]:Aim: our team has confirmed that excessive phosphorylation of tau protein is involved in neuronal escape apoptosis and degenerative death. This study will further study the role of tau protein dephosphorylation in apoptosis and its possible mechanism. Methods: human wild-type tau (tau441) gene was stably transfected into human nephroblastoma cells (HEK293) to establish a stable overexpression of tau cell line: HEK293/tau;. HEK293 cells (HEK293/wt and HEK293/tau) were transiently transfected with plasmid PP2A to overexpression PP2A, transfected with empty pcDNA as control. Then the two kinds of cells were treated with different apoptosis inducer staurosporine,camptothecin,H2O2. After 6 hours of exposure, the apoptosis rate was measured by flow cytometry. The expression and activity of PP2A, anti-apoptotic factor Bcl-2,tau protein were detected by Western blot, and the relationship between the phosphorylation of Bcl-2,tau protein was analyzed. Results: 1) overexpression of PP2A could prevent apoptosis induced by apoptosis inducer. At the same time, the phosphorylation level of ser87 site of anti-apoptotic factor Bcl-2 was significantly decreased. 2) under the action of different apoptosis inducers, the apoptotic rate of HEK293/tau cells with overexpression of tau was significantly lower than that of HEK293/wt cells. When HEK293/tau overexpressed PP2A at the same time, the apoptotic rate increased significantly, while the dephosphorylation of tau protein occurred at ser198/ser199/ser202 site, and the phosphorylation level of anti-apoptotic factor Bcl-2 ser87 site increased significantly. The correlation analysis showed that there was a positive correlation between the dephosphorylation level of ser198/ser199/ser202 site of tau protein and the phosphorylation level of ser87 site of anti-apoptotic factor Bcl-2. Conclusion: overexpression of PP2A can resist apoptosis by dephosphorylation of Bcl-2 at ser87 site. Overexpression of tau protein can resist apoptosis, but when tau protein is dephosphorylated, it may induce apoptosis by upregulating the phosphorylation level of anti-apoptotic factor Bcl-2.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
本文编号：2408970
[Abstract]:Aim: our team has confirmed that excessive phosphorylation of tau protein is involved in neuronal escape apoptosis and degenerative death. This study will further study the role of tau protein dephosphorylation in apoptosis and its possible mechanism. Methods: human wild-type tau (tau441) gene was stably transfected into human nephroblastoma cells (HEK293) to establish a stable overexpression of tau cell line: HEK293/tau;. HEK293 cells (HEK293/wt and HEK293/tau) were transiently transfected with plasmid PP2A to overexpression PP2A, transfected with empty pcDNA as control. Then the two kinds of cells were treated with different apoptosis inducer staurosporine,camptothecin,H2O2. After 6 hours of exposure, the apoptosis rate was measured by flow cytometry. The expression and activity of PP2A, anti-apoptotic factor Bcl-2,tau protein were detected by Western blot, and the relationship between the phosphorylation of Bcl-2,tau protein was analyzed. Results: 1) overexpression of PP2A could prevent apoptosis induced by apoptosis inducer. At the same time, the phosphorylation level of ser87 site of anti-apoptotic factor Bcl-2 was significantly decreased. 2) under the action of different apoptosis inducers, the apoptotic rate of HEK293/tau cells with overexpression of tau was significantly lower than that of HEK293/wt cells. When HEK293/tau overexpressed PP2A at the same time, the apoptotic rate increased significantly, while the dephosphorylation of tau protein occurred at ser198/ser199/ser202 site, and the phosphorylation level of anti-apoptotic factor Bcl-2 ser87 site increased significantly. The correlation analysis showed that there was a positive correlation between the dephosphorylation level of ser198/ser199/ser202 site of tau protein and the phosphorylation level of ser87 site of anti-apoptotic factor Bcl-2. Conclusion: overexpression of PP2A can resist apoptosis by dephosphorylation of Bcl-2 at ser87 site. Overexpression of tau protein can resist apoptosis, but when tau protein is dephosphorylated, it may induce apoptosis by upregulating the phosphorylation level of anti-apoptotic factor Bcl-2.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363
【参考文献】
相关期刊论文 前1条
1 陈晓钎,乌维宁,于常海;14-3-3:保护性信号转导调节蛋白[J];生理科学进展;2004年03期
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