發(fā)布時(shí)間：2019-01-15 07:14

【摘要】： 目的:本課題組已證實(shí)tau蛋白過度磷酸化過程參與了神經(jīng)元逃逸凋亡并發(fā)生退行性變性死亡,本研究將進(jìn)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(jī)制。 方法:先將人最長(zhǎng)的野生型tau(tau441)基因穩(wěn)定轉(zhuǎn)染到人腎母細(xì)胞瘤細(xì)胞(HEK293)內(nèi),以建立穩(wěn)定過度表達(dá)tau的細(xì)胞株:HEK293/tau;再在HEK293細(xì)胞(HEK293/wt和HEK293/tau)中瞬時(shí)轉(zhuǎn)染質(zhì)粒PP2A使之過度表達(dá)PP2A,轉(zhuǎn)染空載體pcDNA作為對(duì)照。接著分別用不同的凋亡誘導(dǎo)劑staurosporine,camptothecin,H2O2處理這兩種細(xì)胞。在作用6小時(shí)后,用流式細(xì)胞計(jì)量術(shù)檢測(cè)細(xì)胞凋亡率;用免疫印跡的技術(shù)檢測(cè)細(xì)胞內(nèi)PP2A、抗凋亡因子Bcl-2、tau蛋白的表達(dá)和活性水平,并分析Bcl-2、tau蛋白磷酸化變化的關(guān)系。 結(jié)果:1)過度表達(dá)PP2A使細(xì)胞可以抵抗凋亡誘導(dǎo)劑所誘導(dǎo)的細(xì)胞凋亡,同時(shí)細(xì)胞內(nèi)抗凋亡因子Bcl-2的ser87位點(diǎn)磷酸化水平顯著下降。2)在不同凋亡誘導(dǎo)劑的作用下:過度表達(dá)tau的HEK293/tau細(xì)胞凋亡率顯著低于HEK293/wt細(xì)胞;當(dāng)HEK293/tau同時(shí)過度表達(dá)PP2A時(shí),凋亡率顯著上升,同時(shí)tau蛋白在ser198/ser199/ser202位點(diǎn)發(fā)生去磷酸化,抗凋亡因子Bcl-2 ser87位點(diǎn)磷酸化水平顯著升高;經(jīng)相關(guān)性分析發(fā)現(xiàn)tau蛋白ser198/ser199/ser202位點(diǎn)的去磷酸化水平與抗凋亡因子Bcl-2的ser87位點(diǎn)磷酸化水平之間存在著正相關(guān)。 結(jié)論:過度表達(dá)PP2A可通過使抗凋亡因子Bcl-2在ser87位點(diǎn)去磷酸化而抵抗細(xì)胞凋亡。過度表達(dá)tau蛋白具有抵抗細(xì)胞凋亡的作用;而當(dāng)tau蛋白被去磷酸化時(shí),可能通過上調(diào)抗凋亡因子Bcl-2磷酸化水平而使細(xì)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(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R363

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