本文關鍵詞： 凋亡 自噬 P62 1 10鄰二氮雜菲雙過氧釩酸鉀 半胱氨酸蛋白酶-1 脫乙�；�-6 微管 焦亡　出處：《安徽醫(yī)科大學》2017年博士論文　論文類型：學位論文

【摘要】：腫瘤是威脅人類健康最嚴重的疾病之一,全世界每天約有2萬人死于腫瘤。目前,腫瘤的發(fā)生機制尚不清楚,也沒有針對腫瘤的特效治療。腫瘤是多因素疾病,不僅與多種遺傳基因相關,還與各種環(huán)境因素相關。Yusuf A Hannun于2016年在《nature》發(fā)表論文表明遺傳因素在腫瘤發(fā)生中只起到10-30%的作用,而環(huán)境因素對腫瘤的發(fā)生影響更大,環(huán)境因素通過影響細胞周期、調節(jié)細胞自噬、干擾正常細胞凋亡、抑制細胞免疫等因素影響腫瘤發(fā)生。自噬是真核生物特有的一種的生命現象,在維持細胞內穩(wěn)態(tài),促進細胞生存方面起重要作用,廣泛參與各種病理生理過程。近些年來,自噬與腫瘤的關系受到廣泛關注,人們期待通過調控自噬干預腫瘤的發(fā)病進程,甚至希望通過調控自噬來徹底治愈腫瘤。腫瘤是慢性的疾病過程,在腫瘤發(fā)生不同階段自噬可能起到不同甚至完全相反的作用。在腫瘤發(fā)生的起始階段,自噬通過改善細胞微環(huán)境抑制腫瘤發(fā)生;然而腫瘤進展階段,自噬可能起到保護腫瘤細胞耐受代謝應激,促進了腫瘤發(fā)展、播散及轉移。但是,自噬對腫瘤的影響是直接作用還是間接作用,調控自噬能否改善腫瘤預后以及如何調控腫瘤自噬問題亟待進一步研究。釩是人體必需微量元素,具有多種生物學活性。在1987年Cantley發(fā)現釩酸根對ATP酶具有抑制作用,繼而科學家又發(fā)現釩酸鹽具有類胰島素樣作用,近年來又發(fā)現釩化合物的抗炎作用、殺精作用。1,10鄰二氮雜菲雙過氧釩酸鉀(BPV(phen))一種穩(wěn)定的過氧釩酸鹽,具有廣泛的生物學活性,Lada Rumora研究表明BPV(phen)能有誘導胰島細胞系RIN-m5F凋亡。Chandler L.Walker研究表明BPV(phen)可能通過抑制調節(jié)細胞自噬夠緩解頸髓損傷。但是BPV(phen)是否具有抗癌活性,是否調節(jié)腫瘤細胞自噬,以及如何調節(jié)細胞自噬機制十分不清。本研究擬探討B(tài)PV(phen)對細胞自噬影響機制,以期為釩化合物用于腫瘤治療提供理論基礎。全部研究內容分為以下四部分:1.BPV(phen)抑制細胞增殖、誘導細胞凋亡和細胞焦亡。探討B(tài)PV(phen)對Hep G2、He LA以及MEF細胞增殖和凋亡的影響。MTT試劑盒檢測BPV(phen)對細胞活力影響,觀察BPV(phen)對細胞形態(tài)學的影響,Western Blotting檢測細胞增殖和凋亡相關蛋白,流式細胞技術檢測細胞凋亡。MTT實驗結果發(fā)現BPV(phen)抑制細胞增殖,LDH活性實驗發(fā)現BPV(phen)誘導細胞釋放LDH,形態(tài)學發(fā)現BPV(phen)誘導細胞形態(tài)逐漸變圓,隨著劑量增大細胞數目減少、部分細胞形態(tài)改變;Western Blotting檢測結果發(fā)現PARP斷裂帶隨劑量依賴性增加,PCNA變化不明顯;Caspase1(對細胞焦亡的形成起決定作用,是細胞焦亡最重要的標志蛋白)呈現劑量依賴性增加。流式細胞學凋亡檢測結果發(fā)現BPV(phen)10μM D2象限細胞明顯增加,凋亡明顯。以上實驗結果提示BPV(phen)抑制細胞增殖,促進細胞凋亡和細胞焦亡。2.BPV(phen)對細胞自噬的影響。鑒于細胞凋亡、細胞焦亡與細胞自噬的密切關系,本研究探討B(tài)PV(phen)對細胞自噬的影響。雌性C57BL/6小鼠適應性飼養(yǎng)1周后,隨機分為兩組,實驗組給予皮下注射BPV(phen)2.5μmol/30 g每天一次×14 d,對照組注射生理鹽水,第15天處死小鼠,取肝臟組織,Western Blotting檢測細胞自噬標志性蛋白LC3。結果發(fā)現BPV(phen)腹腔注射小鼠肝臟組織LC3-Ⅱ表達水平明顯升高,提示自噬啟動被激活或者自噬體降解受到抑制。進一步通過引入自噬體降解抑制劑來闡明BPV(phen)是促進自噬啟動還是抑制自噬體降解。取6孔板培養(yǎng)He La細胞,分為CTRL和BAF組,每組設BPV0、BPV2.5、BPV5、BPV10、BPV20亞組,待細胞生長狀態(tài)良好,各組分別加入BPV(phen)或DMSO至BPV(phen)藥物終濃度分別為0、2.5、5、10、20μM,繼續(xù)培養(yǎng)48 h,在終止細胞培養(yǎng)前12 h,CTRL和BAF組分別加入DMSO或者BAF2μl。收集細胞用Western Blotting實驗檢測LC3蛋白。在Hep G2、MEF細胞重復以上實驗。Western Blotting結果發(fā)現BPV(phen)劑量依賴性增加LC3-Ⅱ的表達,在加入抑制劑后LC3-Ⅱ不隨BPV(phen)變化,在Hep G2、He LA以及MEF細胞系表現相同。在RFP-LC3細胞免疫熒光實驗,觀察10μM BPV(phen)在加入或不加入BAF兩種情況下對RFP-LC3表達的影響。通過共聚焦顯微鏡技術可以觀察到BPV(phen)10M明顯增加RFP-LC3紅色熒光顆粒數目,加入自噬體降解抑制劑BAF后,BPV(phen)組和CTRL組無明顯差別。以上實驗提示,BPV(phen)不是促進自噬的啟動導致自噬體的增多,而是抑制了自噬體的降解而導致自噬體的增多。因此,本研究提示BPV(phen)抑制自噬體的降解。3.BPV(phen)降低P62蛋白水平及其機制。本研究在觀察BPV(phen)對細胞自噬影響時,除了檢測細胞自噬最重要的標志蛋白LC3之外,同時檢測了另一個重要的自噬相關蛋白P62,結果發(fā)現BPV(phen)劑量依賴性降低P62蛋白水平。通常情況下抑制自噬體的降解將會抑制蛋白質通過自噬途徑的降解,表現為P62蛋白水平升高。而本研究發(fā)現BPV(phen)抑制自噬但是P62卻明顯降低,因此BPV(phen)可能是通過其他途徑影響P62蛋白水平。為闡明其機制,He LA細胞系加入蛋白生成抑制劑CHX后進行細胞培養(yǎng),分為BPV(phen)組和CTRL組,觀察在抑制蛋白質合成情況下BPV(phen)對P62水平的影響。結果發(fā)現在抑制蛋白質生成的情況下BPV(phen)降低P62蛋白水平,提示BPV(phen)促進P62的降解。為探明BPV(phen)對P62蛋白水平影響是通過自噬途徑還是蛋白酶體途徑,在He LA細胞系比較在加入自噬抑制劑BAF或和蛋白酶體降解抑制劑MG132情況下BPV(phen)對P62蛋白水平的影響,通過Western Blotting方法檢測P62蛋白水平。結果發(fā)現MG132幾乎逆轉BPV(phen)對P62的降低作用,而應用BAF組僅部分逆轉BPV(phen)對P62的降低作用。此外,取穩(wěn)定表達RFP-LC3的He La細胞系分為三組,加入BPV(phen)培養(yǎng),實驗第48小時收集載玻片對P62進行免疫熒光染色,免疫熒光結果發(fā)現P62與RFP-LC3共定位在BAF應用后出現,而在MG132條件下P62和RFP-LC3沒有共定位。BPV(phen)可能干擾P62和RFP-LC3親和性。為檢測BPV(phen)對P62泛素化水平的影響,在293T細胞過表達P62和Poly-ubiquitin,用Western Blotting檢測分別在有和沒有MG132情況下觀察BPV(phen)對P62和泛素化蛋白的影響。在BPV(phen)作用下表現為非特異性Poly-ubiquitin水平升高。免疫共沉淀實驗發(fā)現,在BPV(phen)作用下泛素化的P62水平增加。本研究提示,BPV(phen)通過增加了P62泛素化水平,促進其通過蛋白酶體途徑降解,從而降低P62蛋白水平。4.BPV(phen)抑制自噬分子機制。為闡明BPV(phen)抑制自噬體降解具體分子機制。通常情況下,自噬體形成后需要通過乙酰化的微管被運送到溶酶體形成自噬溶酶體最終行使物質降解功能,BPV(phen)影響P62穩(wěn)定性與抑制自噬體降解之間可能有某種聯系。在HeLA細胞系沉默P62基因表達,檢測其對對自噬體降解的影響及微管蛋白α-Tubulin乙酰化水平的影響。Western Blotting和免疫熒光檢測結果發(fā)現,P62沉默抑制自噬體的降解,同時α-Tubulin乙酰化水平降低。而在He LA細胞系過表達P62基因,檢測自噬體降解以及α-Tubulin水平,結果與沉默實驗相一致。在He LA細胞系,Western Blotting檢測BPV(phen)對微管蛋白α-Tubulin乙�；降淖兓约癏DAC6蛋白水平的變化。通過免疫共沉淀實驗,檢測P62蛋白水平降低對HDAC6蛋白水平的影響。沉默內源性P62和過表達外源性P62,可以分別抑制和促進自噬體降解。BPV(phen)導致HDAC6與P62相互作用減弱,釋放HDAC6。BPV(phen)通過P62-HDAC6通路抑制自噬體降解。綜上所述,本研究發(fā)現BPV(phen)抑制細胞增殖、促進細胞凋亡和細胞焦亡,同時抑制自噬體降解。其影響自噬體降解的分子通路可能是,BPV(phen)促進P62蛋白酶體降解,后者增強HDAC6活性,繼而HDAC6對乙�；⒐艿鞍椎娜ヒ阴；饔迷黾�,影響自噬體的轉運。
[Abstract]:The tumor is one of the most serious threat to human health, there are about 20 thousand people died of cancer in the world every day. At present, the mechanism of tumorigenesis is not clear, no specific treatment for tumors. The tumor is a multi factor disease, not only related to multiple genes, but also with various ring related environmental factors A Hannun published a paper.Yusuf show that genetic factors in the occurrence of tumor only plays the role of 10-30% in in 2016, and the environmental factors and the occurrence of cancer is more influential environmental factors by influencing the cell cycle, regulation of autophagy, interfere with normal cell apoptosis, inhibition of cell immune factors in tumorigenesis. Autophagy is a unique phenomenon that real life in eukaryotes, cellular homeostasis, promotion plays an important role in cell survival, are involved in various pathophysiological processes. In recent years, the relationship between autophagy and tumor by wide Note, people look through the development process of regulation of autophagy interfere with tumor, even hope that through the regulation of autophagy to completely cure the tumor. The tumor is a chronic disease process in different stages of tumorigenesis, autophagy may play a different role and even completely opposite. In the initial stage of cancer, autophagy by improving cell microenvironment suppresses tumorigenesis; however tumor stage, autophagy may play a protective metabolic stress tolerance of tumor cells, promote tumor development, dissemination and metastasis. But the effect of autophagy on tumor is directly or indirectly, the regulation of autophagy can improve the prognosis of tumor and how to regulate tumor autophagy problems to be studied further. Vanadium is an essential trace element, has a variety of biological activity in 1987. Cantley found that vanadate has an inhibitory effect on ATP enzyme, then scientists found that vanadate with trypsin Insulin like effect, also found recently that the anti-inflammatory effects of vanadium compounds, spermicidal effect.1,10 two o phenanthroline diperoxovanadate acid potassium (BPV (phen)) a stable peroxy vanadate, has extensive biological activities, Lada Rumora study showed that the BPV (phen) can induce islet cell lines RIN-m5F.Chandler L.Walker apoptosis studies showed that BPV (phen) could inhibit the regulation of autophagy alleviated cervical cord injury. But BPV (phen) with anticancer activity, whether and how to regulate tumor cell autophagy, autophagy regulation mechanism is unclear. This study intends to explore the mechanism of BPV (phen) effect on autophagy, in order to to provide a theoretical basis for the treatment of tumors. For all vanadium compound research content is divided into the following four parts: 1.BPV (phen) inhibited cell proliferation, induced apoptosis and pyroptosis. To investigate the BPV (phen) of Hep G2, He LA and MEF cell proliferation and apoptosis .MTT kit to detect the effect of BPV death (phen) effects on cell viability, BPV (phen) to observe the effect on cell morphology, Western Blotting detection of cell proliferation and apoptosis related protein, flow cytometry to detect the apoptosis of.MTT BPV (phen) results showed that the inhibition of cell proliferation, LDH activity test showed that BPV (phen) induced cells to release LDH, BPV (phen) induced morphological cells gradually became round, with the dose increased the number of cells decreased, change cell morphology; Western Blotting showed that with a dose-dependent increase in PARP fault, PCNA did not change significantly; Caspase1 (on the formation of pyroptosis plays a decisive role, is the cell the most important sign of focal dead protein) in a dose-dependent manner. Flow cytometry apoptosis detection results showed that BPV (phen) M 10 quadrant D2 cells increased and apoptosis significantly. The above results suggest that BPV (pH EN) inhibited cell proliferation, promote cell apoptosis and pyroptosis.2.BPV (phen) effect on autophagy. Due to apoptosis, pyroptosis close relationship with autophagy, in this study, BPV (phen) effect on autophagy. Female C57BL/6 mice were fed for 1 weeks, were randomly divided into two groups, the experimental group received subcutaneous injection of BPV (phen) 2.5 mol/30 g * 14 d once a day, the control group were injected with saline for fifteenth days, the mice were killed, the liver tissue, Western Blotting detecting autophagy marker protein LC3. results showed that BPV (phen) expression in liver tissue of mice peritoneal injection of LC3- II level was significantly increased, suggesting that autophagy is activated or inhibited autophagy degradation. Further through the introduction of autophagy inhibitors to elucidate the degradation of BPV (phen) is to promote or inhibit autophagy autophagy degradation. 6 Kong Banpei had He La cells, divided into CTRL and BA F group, each group with BPV0, BPV2.5, BPV5, BPV10, BPV20 subgroups, the cells grew well and were joined BPV (phen or DMSO) to BPV (phen) concentration were 0,2.5,5,10,20 M, cultured for 48 h, at the end of cell culture before 12 h, CTRL and BAF components don't join DMSO or BAF2 L. cells were collected with LC3 Western protein was detected by Blotting experiment. In Hep G2 MEF cells, repeat the above experiment results showed that BPV.Western Blotting (phen) dose dependently increased the expression of LC3- II, before joining LC3- II with BPV inhibitor (phen) in Hep G2, He, and LA MEF cell lines showed the same. In RFP-LC3 immunofluorescence experiments, observation of 10 M BPV (phen) effect on the expression of RFP-LC3 in BAF is added or not under two conditions. By confocal microscopy can be observed in the BPV (phen) 10M significantly increased the number of RFP-LC3 red fluorescent particles, adding autophagy The biodegradation inhibitor BAF, BPV (phen) showed no significant difference between the group and CTRL group. The above experiments suggest that BPV (phen) is to promote autophagy start leads to increased autophagy, but inhibited the degradation of autophagy and leads to increased autophagy. Therefore, this study suggests that BPV (phen).3.BPV degradation and inhibition of autophagy the lower level of P62 protein (phen) and its mechanism. In this study, observation of BPV (phen) on autophagy effect, in addition to the most important detecting autophagy marker protein LC3, also detected another important autophagy related protein P62, the results showed that BPV (phen) dose dependently reduced P62 protein level the degradation will normally inhibit. Autophagy inhibition of protein degradation by autophagy pathway, increased P62 protein levels. The study found that BPV (phen) inhibition of autophagy but P62 was significantly reduced, so the BPV (phen) may be through the other Affect the protein level of P62. To elucidate the mechanism of He LA cell line into protein inhibitor CHX after cell culture, divided into BPV (phen) group and CTRL group were observed in the inhibition of protein synthesis by BPV (phen) effect on P62 level. The results found in the inhibition of protein generated by BPV (phen) P62 protein levels were reduced, suggesting that BPV (phen) to promote the degradation of P62. BPV (phen) to explore the influence on the level of P62 protein through the autophagy pathway or proteasome pathway, in He LA cell lines in autophagy inhibitor BAF or MG132 inhibitor and proteasome degradation under the condition of BPV (phen) effect on the protein level of P62, through the Western Blotting method to detect the protein level of P62. The results showed that MG132 BPV (phen) almost reversed the decrease in P62, and the application of group BAF was only partially reversed BPV (phen) of P62 decreased. In addition, the stable expression of RFP-LC3 The He La cell lines were divided into three groups, adding BPV (phen) training, forty-eighth hours to collect experimental slides for P62 immunofluorescence staining, immunofluorescence showed that P62 colocalized with RFP-LC3 in BAF application, and under the condition of MG132 P62 and RFP-LC3 have no co localization of.BPV (phen) and may interfere with P62 RFP-LC3 affinity. For the detection of BPV (phen) effect on P62 ubiquitination level, overexpression of P62 and Poly-ubiquitin in 293T cells by Western, Blotting and BPV were detected in the observation of no MG132 case (phen) effects on P62 and ubiquitin proteins. In BPV (phen) under the action of increased non the specific Poly-ubiquitin level. Co immunoprecipitation experiments revealed that in BPV (phen) P62 level of ubiquitination under the action of increasing. This study suggested that BPV (phen) by increasing P62 ubiquitination level, promote the degradation through the proteasome pathway, thereby reducing the level of P62 protein .4.BPV (phen) in order to elucidate the molecular mechanism of inhibition of autophagy. BPV (phen) inhibition of autophagy specific degradation mechanism. Under normal circumstances, after the formation of autophagosomes through acetylated microtubules are transported to lysosomes to form autolysosome final exercise material degradation function, BPV (phen) may have some relationship between the stability and the inhibition effect of P62 autophagic degradation. Expression of P62 gene silencing in HeLA cells, detect the effect on autophagy degradation and alpha tubulin acetylation level of -Tubulin the effect of.Western Blotting and immunofluorescence test results showed that the degradation of P62 silencing inhibited autophagy, while alpha acetylation level of -Tubulin decreased. Over expression of P62 gene in He LA cell line, detect autophagosome degradation and alpha -Tubulin level. The results are consistent with the experimental silence. In He LA cell line, Western Blotting BPV (phen) detection of micro tube protein alpha -Tubulin The change of histone acetylation and HDAC6 protein level changes. By CO immunoprecipitation assay to detect the protein level of P62, reduce the impact on the level of HDAC6 protein. The silencing of endogenous P62 and overexpression of exogenous P62, can inhibit and promote autophagic degradation of.BPV (phen) in HDAC6 and P62 interaction is weakened, the release of HDAC6.BPV (phen inhibition of autophagy by P62-HDAC6) degradation pathway. In summary, this study found that BPV (phen) inhibited cell proliferation, promote cell apoptosis and pyroptosis, while inhibiting autophagy degradation. The influence of molecular pathways of autophagy may be degradation, BPV (phen) P62 promote proteasome degradation, which enhance the activity of HDAC6, and HDAC6 the acetylated tubulin acetylation increased influence of autophagosome transport.

【學位授予單位】：安徽醫(yī)科大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R96

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8 陳永;鄒s舠，

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