本文選題：自噬 + 自噬流�。� 參考：《北京協(xié)和醫(yī)學(xué)院》2015年博士論文

【摘要】：自噬是細(xì)胞內(nèi)的一種分解代謝過(guò)程,用于維持細(xì)胞內(nèi)物質(zhì)及能量的穩(wěn)態(tài)。正常生理狀態(tài)下,自噬常維持在基礎(chǔ)水平,而在腫瘤、糖尿病、炎癥及神經(jīng)退行性疾病等多種病理狀態(tài)下往往都伴隨有自噬活性的改變。實(shí)驗(yàn)室前期研究也證實(shí)腫瘤、纖維化疾病及代謝綜合癥中均存在自噬活性的失調(diào),我們認(rèn)為,篩選不同活性的自噬調(diào)節(jié)劑應(yīng)用于以上疾病模型具有治療意義。因此,我們建立了自噬活性物質(zhì)篩選系統(tǒng),從具有結(jié)構(gòu)及功能多樣性的天然產(chǎn)物化合物庫(kù)中篩選到多個(gè)自噬激動(dòng)劑及自噬抑制劑,其中以XY1為代表的黃烷類(lèi)化合物及以ZH33為代表的二氫黃酮類(lèi)化合物能夠明顯激活自噬流。較強(qiáng)的自噬激活能夠?qū)е伦允尚约?xì)胞死亡,而誘導(dǎo)腫瘤細(xì)胞發(fā)生自噬性細(xì)胞死亡是克服凋亡耐受的有效途徑。自噬激動(dòng)劑XY1及ZH33均具有抗腫瘤活性。XY1對(duì)小鼠黑色素瘤細(xì)胞B16-F10具有較強(qiáng)的抑制作用,而對(duì)小鼠AML-12正常肝細(xì)胞影響較小。進(jìn)一步分子機(jī)制的研究發(fā)現(xiàn),XY1可以選擇性增加腫瘤細(xì)胞內(nèi)ROS水平,對(duì)正常細(xì)胞無(wú)影響。利用NAC阻斷細(xì)胞內(nèi)ROS的產(chǎn)生及敲除Atg5抑制細(xì)胞自噬活性均可以減少XY1誘導(dǎo)的腫瘤細(xì)胞凋亡,說(shuō)明XY1的抗腫瘤活性是自噬激活及ROS增加的共同作用結(jié)果。動(dòng)物實(shí)驗(yàn)也表明,XY1可以抑制小鼠黑色素瘤的生長(zhǎng),激活腫瘤組織自噬,選擇性誘導(dǎo)腫瘤組織ROS堆積。ZH33對(duì)人肝癌細(xì)胞Hepg2具有一定的抑制作用,分子機(jī)制研究結(jié)果顯示ZH33也可以增加腫瘤細(xì)胞內(nèi)ROS水平,誘導(dǎo)腫瘤細(xì)胞發(fā)生非凋亡依賴(lài)的自噬性細(xì)胞死亡。糖尿病是嚴(yán)重威脅人類(lèi)健康的代謝紊亂疾病,而糖尿病心肌病作為糖尿病的主要并發(fā)癥,是導(dǎo)致糖尿病患者死亡的首要原因。自噬作為一種物質(zhì)及能量代謝過(guò)程與糖尿病的發(fā)生發(fā)展密切相關(guān),目前對(duì)于自噬在2型糖尿病心肌病中的作用仍不明了。本研究利用高脂膳食合并STZ方法制備小鼠2型糖尿病心肌病模型,并從自噬流的角度發(fā)現(xiàn)2型糖尿病心肌病中自噬過(guò)度活化,表現(xiàn)為通過(guò)上游PI3K/AKT-mTOR及AMPK-mTOR信號(hào)通路激活自噬,自噬核心復(fù)合物PI3KC3、 Beclinl表達(dá)增加,自噬標(biāo)志蛋白LC3-Ⅱ增加,自噬貨車(chē)蛋白P62、NBR1及NIX下降。ERBB信號(hào)通路對(duì)于維持心肌細(xì)胞正常生長(zhǎng)及損傷修復(fù)非常重要,我們發(fā)現(xiàn)伴隨著心肌細(xì)胞自噬活化,ERBB家族蛋白降解增加導(dǎo)致ERBB信號(hào)通路受損。綜上,利用自噬活性物質(zhì)篩選系統(tǒng)我們篩選到了多種自噬激動(dòng)劑及抑制劑,為自噬相關(guān)疾病的治療提供了物質(zhì)基礎(chǔ)。一方面,我們通過(guò)對(duì)XY1及ZH33這兩種自噬激動(dòng)劑抗腫瘤作用及分子機(jī)制的研究,為自噬激動(dòng)劑應(yīng)用于抗腫瘤藥物開(kāi)發(fā)提供了理論依據(jù)。另一方面,我們通過(guò)闡明自噬激活促進(jìn)糖尿病心肌病發(fā)生發(fā)展的分子機(jī)制,為自噬抑制劑應(yīng)用于糖尿病心肌病治療提供了新的理論依據(jù)。
[Abstract]:Autophagy is a catabolism process in cells, which is used to maintain the homeostasis of substance and energy in cells. In normal physiological state autophagy is often maintained at the basic level while autophagy is often accompanied by changes in autophagy in various pathological states such as tumor diabetes inflammation and neurodegenerative diseases. Previous laboratory studies have also confirmed that autophagy disorders exist in tumors, fibrotic diseases and metabolic syndrome. We believe that screening different active autophagy modulators for the above disease models has therapeutic significance. Therefore, we have established a screening system for autophagy active substances. A number of autophagy agonists and autophagy inhibitors have been screened from natural product compounds with structural and functional diversity. Among them, flavanides represented by XY1 and dihydroflavonoids represented by ZH33 could activate autophagy obviously. Strong autophagy activation can lead to autophagy cell death, and inducing autophagic cell death is an effective way to overcome apoptosis tolerance. Both autophagy agonists XY1 and ZH33 have anti-tumor activity. XY1 has a strong inhibitory effect on B16-F10 of mouse melanoma cells, but has little effect on normal hepatocytes of mice AML-12. Further molecular studies showed that XY1 could selectively increase the level of ROS in tumor cells, but had no effect on normal cells. Blocking the production of ROS and knockout of Atg5 by NAC can reduce the apoptosis of tumor cells induced by XY1, indicating that the anti-tumor activity of XY1 is the result of both autophagy activation and ROS increase. Animal experiments also showed that XY1 could inhibit the growth of murine melanoma, activate tumor autophagy, and selectively induce tumor tissue ROS accumulation. ZH33 could inhibit the Hepg2 of human hepatoma cells. The results of molecular mechanism showed that ZH33 could also increase the level of ROS in tumor cells and induce apoptosis dependent autophagy cell death. Diabetes mellitus (DM) is a metabolic disorder that threatens human health seriously. As a major complication of diabetes, diabetic cardiomyopathy (DM) is the leading cause of death in diabetic patients. Autophagy as a metabolic process of substance and energy is closely related to the occurrence and development of diabetes mellitus. The role of autophagy in type 2 diabetic cardiomyopathy is still unclear. In this study, the model of type 2 diabetic cardiomyopathy in mice was established by high fat diet combined with STZ method. From the point of view of autophagy, it was found that autophagy was activated by upstream PI3K/AKT-mTOR and AMPK-mTOR signaling pathway in type 2 diabetic cardiomyopathy. The expression of PI3KC3, Beclinl and LC3- 鈪，

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