【摘要】： 目的:本課題組已證實tau蛋白過度磷酸化過程參與了神經元逃逸凋亡并發(fā)生退行性變性死亡,本研究將進一步研究tau蛋白去磷酸化過程在凋亡發(fā)生中的作用及可能機制。 方法:先將人最長的野生型tau(tau441)基因穩(wěn)定轉染到人腎母細胞瘤細胞(HEK293)內,以建立穩(wěn)定過度表達tau的細胞株:HEK293/tau;再在HEK293細胞(HEK293/wt和HEK293/tau)中瞬時轉染質粒PP2A使之過度表達PP2A,轉染空載體pcDNA作為對照。接著分別用不同的凋亡誘導劑staurosporine,camptothecin,H2O2處理這兩種細胞。在作用6小時后,用流式細胞計量術檢測細胞凋亡率;用免疫印跡的技術檢測細胞內PP2A、抗凋亡因子Bcl-2、tau蛋白的表達和活性水平,并分析Bcl-2、tau蛋白磷酸化變化的關系。 結果:1)過度表達PP2A使細胞可以抵抗凋亡誘導劑所誘導的細胞凋亡,同時細胞內抗凋亡因子Bcl-2的ser87位點磷酸化水平顯著下降。2)在不同凋亡誘導劑的作用下:過度表達tau的HEK293/tau細胞凋亡率顯著低于HEK293/wt細胞;當HEK293/tau同時過度表達PP2A時,凋亡率顯著上升,同時tau蛋白在ser198/ser199/ser202位點發(fā)生去磷酸化,抗凋亡因子Bcl-2 ser87位點磷酸化水平顯著升高;經相關性分析發(fā)現(xiàn)tau蛋白ser198/ser199/ser202位點的去磷酸化水平與抗凋亡因子Bcl-2的ser87位點磷酸化水平之間存在著正相關。 結論:過度表達PP2A可通過使抗凋亡因子Bcl-2在ser87位點去磷酸化而抵抗細胞凋亡。過度表達tau蛋白具有抵抗細胞凋亡的作用;而當tau蛋白被去磷酸化時,可能通過上調抗凋亡因子Bcl-2磷酸化水平而使細胞發(fā)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.
【學位授予單位】：華中科技大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R363

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1 陳曉釬,烏維寧,于常海;14-3-3:保護性信號轉導調節(jié)蛋白[J];生理科學進展;2004年03期

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本文編號：2408970