抗凋亡蛋白Bcl-2在自噬调控以及细胞生存中的机制研究

发布时间：2018-03-29 09:36

本文选题：Bcl-2　切入点：自噬　出处：《苏州大学》2012年博士论文

【摘要】：目的：抗凋亡蛋白Bcl-2在急性饥饿时，通过与自噬关键蛋白Beclin1的相互作用，抑制Beclin1诱导的自噬，但是在某些情况下，Bcl-2的过表达并不能抑制自噬活性，因此抗凋亡蛋白Bcl-2和自噬之间的关系有待进一步研究。本研究的目的是探讨抗凋亡蛋白Bcl-2在自噬调控中的作用及其在细胞生存和死亡中的意义。方法：利用血清饥饿和自噬激动剂雷帕霉素（Rapamycin）在人神经母细胞瘤SH-SY5Y中，激活自噬，Western检测自噬相关蛋白Beclin1，LC3，p62和CathepsinD，确定自噬激活模型成功；检测Bcl-2家族蛋白变化；利用siRNA Knockdown Bcl-2，或者应用Bcl-2的小分子抑制剂拮抗Bcl-2的功能，用MTT法检测细胞在饥饿下的活力变化，用Annexin-V/PI检测细胞凋亡的改变；加入自噬不同阶段的抑制剂，3-MA，E64D和Bafilomycin A1，检测在Bcl-2被下调或被小分子抑制剂拮抗时，血清饥饿诱导的自噬对细胞活力的影响。结果：利用血清饥饿SH-SY5Y细胞6，12，24，36，48小时，Western检测自噬蛋白Beclin1，LC3，Cathepsin D和p62，结果显示Beclin1，LC3和Cathepsin D在血清饥饿时均上调，而自噬底物p62在6小时一过性下调，提示自噬被激活；用自噬激动剂雷帕霉素同样激活了自噬；在自噬被激活时，检测到抗凋亡蛋白Bcl-2上调；用siRNA干扰Bcl-2蛋白表达水平，检测到LC3无变化，加入溶酶体抑制剂氯化铵，，LC3出现堆积，并且自噬底物p62被进一步降解，提示自噬流量进一步增强；在Bcl-2被下调时，或者应用小分子抑制剂HA14-1和ABT-737拮抗Bcl-2的功能活性时，MTT结果显示，血清饥饿进一步使细胞活力下降，Annexin V/PI结果显示细胞发生早期和晚期凋亡；泛caspase抑制剂Z-VAD阻断细胞死亡证实部分细胞死亡是凋亡性的；应用自噬不同阶段的抑制剂，E64D和巴普洛霉素（Bafilomycin A1）抑制自噬，可以挽救血清饥饿导致的部分细胞死亡。这些结果表明，伴随自噬激活而上调的Bcl-2蛋白能够限制自噬的过度激活和保护营养应激下的细胞死亡，当Bcl-2被下调时，自噬进一步激活，并且细胞发生死亡，这种死亡是凋亡性的，同时又是自噬性，因为caspase的抑制剂和自噬的抑制剂都能够挽救部分细胞死亡。结论：营养剥夺诱导的自噬激活在生理条件下是保护性的，当Bcl-2下调时，抗凋亡蛋白与促凋亡蛋白，抗自噬蛋白与促自噬蛋白之间的力量对比发生变化，这时候，上调自噬活性可以导致营养应激条件下的细胞死亡,提示自噬的保护性作用依赖于抗凋亡蛋白Bcl-2，一旦Bcl-2下调或者功能受损，自噬的激活对细胞死亡有贡献。
[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.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

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