本文選題：Bcl-2　切入點(diǎn)：自噬　出處：《蘇州大學(xué)》2012年博士論文

【摘要】：目的：抗凋亡蛋白Bcl-2在急性饑餓時(shí)，通過與自噬關(guān)鍵蛋白Beclin1的相互作用，抑制Beclin1誘導(dǎo)的自噬，但是在某些情況下，Bcl-2的過表達(dá)并不能抑制自噬活性，因此抗凋亡蛋白Bcl-2和自噬之間的關(guān)系有待進(jìn)一步研究。本研究的目的是探討抗凋亡蛋白Bcl-2在自噬調(diào)控中的作用及其在細(xì)胞生存和死亡中的意義。 方法：利用血清饑餓和自噬激動(dòng)劑雷帕霉素（Rapamycin）在人神經(jīng)母細(xì)胞瘤SH-SY5Y中，激活自噬，Western檢測(cè)自噬相關(guān)蛋白Beclin1，LC3，p62和CathepsinD，確定自噬激活模型成功；檢測(cè)Bcl-2家族蛋白變化；利用siRNA Knockdown Bcl-2，或者應(yīng)用Bcl-2的小分子抑制劑拮抗Bcl-2的功能，用MTT法檢測(cè)細(xì)胞在饑餓下的活力變化，用Annexin-V/PI檢測(cè)細(xì)胞凋亡的改變；加入自噬不同階段的抑制劑，3-MA，E64D和Bafilomycin A1，檢測(cè)在Bcl-2被下調(diào)或被小分子抑制劑拮抗時(shí)，血清饑餓誘導(dǎo)的自噬對(duì)細(xì)胞活力的影響。 結(jié)果：利用血清饑餓SH-SY5Y細(xì)胞6，12，24，36，48小時(shí)，Western檢測(cè)自噬蛋白Beclin1，LC3，Cathepsin D和p62，結(jié)果顯示Beclin1，LC3和Cathepsin D在血清饑餓時(shí)均上調(diào)，而自噬底物p62在6小時(shí)一過性下調(diào)，提示自噬被激活；用自噬激動(dòng)劑雷帕霉素同樣激活了自噬；在自噬被激活時(shí)，檢測(cè)到抗凋亡蛋白Bcl-2上調(diào)；用siRNA干擾Bcl-2蛋白表達(dá)水平，檢測(cè)到LC3無變化，加入溶酶體抑制劑氯化銨，，LC3出現(xiàn)堆積，并且自噬底物p62被進(jìn)一步降解，提示自噬流量進(jìn)一步增強(qiáng)；在Bcl-2被下調(diào)時(shí)，或者應(yīng)用小分子抑制劑HA14-1和ABT-737拮抗Bcl-2的功能活性時(shí)，MTT結(jié)果顯示，血清饑餓進(jìn)一步使細(xì)胞活力下降，Annexin V/PI結(jié)果顯示細(xì)胞發(fā)生早期和晚期凋亡；泛caspase抑制劑Z-VAD阻斷細(xì)胞死亡證實(shí)部分細(xì)胞死亡是凋亡性的；應(yīng)用自噬不同階段的抑制劑，E64D和巴普洛霉素（Bafilomycin A1）抑制自噬，可以挽救血清饑餓導(dǎo)致的部分細(xì)胞死亡。這些結(jié)果表明，伴隨自噬激活而上調(diào)的Bcl-2蛋白能夠限制自噬的過度激活和保護(hù)營養(yǎng)應(yīng)激下的細(xì)胞死亡，當(dāng)Bcl-2被下調(diào)時(shí)，自噬進(jìn)一步激活，并且細(xì)胞發(fā)生死亡，這種死亡是凋亡性的，同時(shí)又是自噬性，因?yàn)閏aspase的抑制劑和自噬的抑制劑都能夠挽救部分細(xì)胞死亡。 結(jié)論：營養(yǎng)剝奪誘導(dǎo)的自噬激活在生理?xiàng)l件下是保護(hù)性的，當(dāng)Bcl-2下調(diào)時(shí)，抗凋亡蛋白與促凋亡蛋白，抗自噬蛋白與促自噬蛋白之間的力量對(duì)比發(fā)生變化，這時(shí)候，上調(diào)自噬活性可以導(dǎo)致營養(yǎng)應(yīng)激條件下的細(xì)胞死亡,提示自噬的保護(hù)性作用依賴于抗凋亡蛋白Bcl-2，一旦Bcl-2下調(diào)或者功能受損，自噬的激活對(duì)細(xì)胞死亡有貢獻(xiàn)。
[Abstract]:Objective: during acute starvation, anti-apoptotic protein Bcl-2 inhibited autophagy induced by Beclin1 by interacting with autophagy key protein Beclin1, but in some cases, the overexpression of Bcl-2 could not inhibit autophagy activity. Therefore, the relationship between anti-apoptotic protein Bcl-2 and autophagy needs further study. The purpose of this study is to investigate the role of anti-apoptotic protein Bcl-2 in autophagy regulation and its significance in cell survival and death. Methods: in human neuroblastoma SH-SY5Y, serum starvation and autophagy agonist rapamycinin were used to activate autophagy associated proteins Beclin1, LC3, p62 and CathepsinD, to determine the success of autophagy activation model and to detect the changes of Bcl-2 family proteins. Using siRNA Knockdown Bcl-2 or Bcl-2 small molecule inhibitor to antagonize the function of Bcl-2, the changes of cell viability under starvation and apoptosis were detected by MTT and Annexin-V/PI respectively. The effects of serum starvation induced autophagy on cell viability were detected when Bcl-2 was down-regulated or antagonized by small molecular inhibitors. Results: the autophagy protein Beclin1LC3L ~ (3 +) Cathepsin D and p62D were detected in SH-SY5Y cells with serum starvation by Western blot. The results showed that both Beclin1LC _ 3 and Cathepsin _ D were up-regulated during serum starvation, while the autophagy substrate p62 was down-regulated at 6 hours, suggesting that autophagy was activated. Autophagic agonist rapamycin also activated autophagy; when autophagy was activated, anti-apoptotic protein Bcl-2 was up-regulated; siRNA interfered with the expression of Bcl-2 protein, detected no change in LC3, and accumulated in lysosomal inhibitor ammonium chloride LC3. Moreover, autophagy substrate p62 was further degraded, suggesting a further increase in autophagy flow, or when Bcl-2 was down-regulated, or when HA14-1 and ABT-737 were used to antagonize the functional activity of Bcl-2. The results of Annexin V/PI showed that cell apoptosis occurred in early and late stages, and that Z-VAD, a pan-#en0# inhibitor, blocked cell death and confirmed that some cell death was apoptotic. Inhibition of autophagy by different stages of autophagy, such as E64D and Bafilomycin A1, can save some of the cell death caused by serum starvation. The up-regulated Bcl-2 protein associated with autophagy can limit the excessive activation of autophagy and protect cell death under nutritional stress. When Bcl-2 is down-regulated, autophagy is further activated and the cells die, which is apoptotic. It is also autophagy because both caspase inhibitors and autophagy inhibitors can save some cell death. Conclusion: the activation of autophagy induced by nutrition deprivation is protective under physiological conditions. When Bcl-2 is down-regulated, the power contrast between anti-apoptotic protein and pro-apoptotic protein, anti-autophagy protein and autophagy protein is changed. Upregulating autophagy activity can lead to cell death under nutritional stress, suggesting that the protective effect of autophagy depends on anti-apoptotic protein Bcl-2. Once Bcl-2 is down-regulated or function impaired, autophagy activation contributes to cell death.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

【共引文獻(xiàn)】

相關(guān)博士學(xué)位論文 前1條

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相關(guān)碩士學(xué)位論文 前1條

1 郭旭光;肺泡II型上皮細(xì)胞自噬體在結(jié)核分枝桿菌感染過程中作用的研究[D];第四軍醫(yī)大學(xué);2010年

本文編號(hào)：1680558