1,10邻二氮杂菲双过氧钒酸钾对细胞自噬的影响及其机制研究

发布时间：2018-01-22 08:22

本文关键词： 凋亡自噬 P62 1 10邻二氮杂菲双过氧钒酸钾半胱氨酸蛋白酶-1 脱乙酰化酶-6 微管焦亡　出处：《安徽医科大学》2017年博士论文　论文类型：学位论文

【摘要】：肿瘤是威胁人类健康最严重的疾病之一,全世界每天约有2万人死于肿瘤。目前,肿瘤的发生机制尚不清楚,也没有针对肿瘤的特效治疗。肿瘤是多因素疾病,不仅与多种遗传基因相关,还与各种环境因素相关。Yusuf A Hannun于2016年在《nature》发表论文表明遗传因素在肿瘤发生中只起到10-30%的作用,而环境因素对肿瘤的发生影响更大,环境因素通过影响细胞周期、调节细胞自噬、干扰正常细胞凋亡、抑制细胞免疫等因素影响肿瘤发生。自噬是真核生物特有的一种的生命现象,在维持细胞内稳态,促进细胞生存方面起重要作用,广泛参与各种病理生理过程。近些年来,自噬与肿瘤的关系受到广泛关注,人们期待通过调控自噬干预肿瘤的发病进程,甚至希望通过调控自噬来彻底治愈肿瘤。肿瘤是慢性的疾病过程,在肿瘤发生不同阶段自噬可能起到不同甚至完全相反的作用。在肿瘤发生的起始阶段,自噬通过改善细胞微环境抑制肿瘤发生;然而肿瘤进展阶段,自噬可能起到保护肿瘤细胞耐受代谢应激,促进了肿瘤发展、播散及转移。但是,自噬对肿瘤的影响是直接作用还是间接作用,调控自噬能否改善肿瘤预后以及如何调控肿瘤自噬问题亟待进一步研究。钒是人体必需微量元素,具有多种生物学活性。在1987年Cantley发现钒酸根对ATP酶具有抑制作用,继而科学家又发现钒酸盐具有类胰岛素样作用,近年来又发现钒化合物的抗炎作用、杀精作用。1,10邻二氮杂菲双过氧钒酸钾(BPV(phen))一种稳定的过氧钒酸盐,具有广泛的生物学活性,Lada Rumora研究表明BPV(phen)能有诱导胰岛细胞系RIN-m5F凋亡。Chandler L.Walker研究表明BPV(phen)可能通过抑制调节细胞自噬够缓解颈髓损伤。但是BPV(phen)是否具有抗癌活性,是否调节肿瘤细胞自噬,以及如何调节细胞自噬机制十分不清。本研究拟探讨BPV(phen)对细胞自噬影响机制,以期为钒化合物用于肿瘤治疗提供理论基础。全部研究内容分为以下四部分:1.BPV(phen)抑制细胞增殖、诱导细胞凋亡和细胞焦亡。探讨BPV(phen)对Hep G2、He LA以及MEF细胞增殖和凋亡的影响。MTT试剂盒检测BPV(phen)对细胞活力影响,观察BPV(phen)对细胞形态学的影响,Western Blotting检测细胞增殖和凋亡相关蛋白,流式细胞技术检测细胞凋亡。MTT实验结果发现BPV(phen)抑制细胞增殖,LDH活性实验发现BPV(phen)诱导细胞释放LDH,形态学发现BPV(phen)诱导细胞形态逐渐变圆,随着剂量增大细胞数目减少、部分细胞形态改变;Western Blotting检测结果发现PARP断裂带随剂量依赖性增加,PCNA变化不明显;Caspase1(对细胞焦亡的形成起决定作用,是细胞焦亡最重要的标志蛋白)呈现剂量依赖性增加。流式细胞学凋亡检测结果发现BPV(phen)10μM D2象限细胞明显增加,凋亡明显。以上实验结果提示BPV(phen)抑制细胞增殖,促进细胞凋亡和细胞焦亡。2.BPV(phen)对细胞自噬的影响。鉴于细胞凋亡、细胞焦亡与细胞自噬的密切关系,本研究探讨BPV(phen)对细胞自噬的影响。雌性C57BL/6小鼠适应性饲养1周后,随机分为两组,实验组给予皮下注射BPV(phen)2.5μmol/30 g每天一次×14 d,对照组注射生理盐水,第15天处死小鼠,取肝脏组织,Western Blotting检测细胞自噬标志性蛋白LC3。结果发现BPV(phen)腹腔注射小鼠肝脏组织LC3-Ⅱ表达水平明显升高,提示自噬启动被激活或者自噬体降解受到抑制。进一步通过引入自噬体降解抑制剂来阐明BPV(phen)是促进自噬启动还是抑制自噬体降解。取6孔板培养He La细胞,分为CTRL和BAF组,每组设BPV0、BPV2.5、BPV5、BPV10、BPV20亚组,待细胞生长状态良好,各组分别加入BPV(phen)或DMSO至BPV(phen)药物终浓度分别为0、2.5、5、10、20μM,继续培养48 h,在终止细胞培养前12 h,CTRL和BAF组分别加入DMSO或者BAF2μl。收集细胞用Western Blotting实验检测LC3蛋白。在Hep G2、MEF细胞重复以上实验。Western Blotting结果发现BPV(phen)剂量依赖性增加LC3-Ⅱ的表达,在加入抑制剂后LC3-Ⅱ不随BPV(phen)变化,在Hep G2、He LA以及MEF细胞系表现相同。在RFP-LC3细胞免疫荧光实验,观察10μM BPV(phen)在加入或不加入BAF两种情况下对RFP-LC3表达的影响。通过共聚焦显微镜技术可以观察到BPV(phen)10M明显增加RFP-LC3红色荧光颗粒数目,加入自噬体降解抑制剂BAF后,BPV(phen)组和CTRL组无明显差别。以上实验提示,BPV(phen)不是促进自噬的启动导致自噬体的增多,而是抑制了自噬体的降解而导致自噬体的增多。因此,本研究提示BPV(phen)抑制自噬体的降解。3.BPV(phen)降低P62蛋白水平及其机制。本研究在观察BPV(phen)对细胞自噬影响时,除了检测细胞自噬最重要的标志蛋白LC3之外,同时检测了另一个重要的自噬相关蛋白P62,结果发现BPV(phen)剂量依赖性降低P62蛋白水平。通常情况下抑制自噬体的降解将会抑制蛋白质通过自噬途径的降解,表现为P62蛋白水平升高。而本研究发现BPV(phen)抑制自噬但是P62却明显降低,因此BPV(phen)可能是通过其他途径影响P62蛋白水平。为阐明其机制,He LA细胞系加入蛋白生成抑制剂CHX后进行细胞培养,分为BPV(phen)组和CTRL组,观察在抑制蛋白质合成情况下BPV(phen)对P62水平的影响。结果发现在抑制蛋白质生成的情况下BPV(phen)降低P62蛋白水平,提示BPV(phen)促进P62的降解。为探明BPV(phen)对P62蛋白水平影响是通过自噬途径还是蛋白酶体途径,在He LA细胞系比较在加入自噬抑制剂BAF或和蛋白酶体降解抑制剂MG132情况下BPV(phen)对P62蛋白水平的影响,通过Western Blotting方法检测P62蛋白水平。结果发现MG132几乎逆转BPV(phen)对P62的降低作用,而应用BAF组仅部分逆转BPV(phen)对P62的降低作用。此外,取稳定表达RFP-LC3的He La细胞系分为三组,加入BPV(phen)培养,实验第48小时收集载玻片对P62进行免疫荧光染色,免疫荧光结果发现P62与RFP-LC3共定位在BAF应用后出现,而在MG132条件下P62和RFP-LC3没有共定位。BPV(phen)可能干扰P62和RFP-LC3亲和性。为检测BPV(phen)对P62泛素化水平的影响,在293T细胞过表达P62和Poly-ubiquitin,用Western Blotting检测分别在有和没有MG132情况下观察BPV(phen)对P62和泛素化蛋白的影响。在BPV(phen)作用下表现为非特异性Poly-ubiquitin水平升高。免疫共沉淀实验发现,在BPV(phen)作用下泛素化的P62水平增加。本研究提示,BPV(phen)通过增加了P62泛素化水平,促进其通过蛋白酶体途径降解,从而降低P62蛋白水平。4.BPV(phen)抑制自噬分子机制。为阐明BPV(phen)抑制自噬体降解具体分子机制。通常情况下,自噬体形成后需要通过乙酰化的微管被运送到溶酶体形成自噬溶酶体最终行使物质降解功能,BPV(phen)影响P62稳定性与抑制自噬体降解之间可能有某种联系。在HeLA细胞系沉默P62基因表达,检测其对对自噬体降解的影响及微管蛋白α-Tubulin乙酰化水平的影响。Western Blotting和免疫荧光检测结果发现,P62沉默抑制自噬体的降解,同时α-Tubulin乙酰化水平降低。而在He LA细胞系过表达P62基因,检测自噬体降解以及α-Tubulin水平,结果与沉默实验相一致。在He LA细胞系,Western Blotting检测BPV(phen)对微管蛋白α-Tubulin乙酰化水平的变化以及HDAC6蛋白水平的变化。通过免疫共沉淀实验,检测P62蛋白水平降低对HDAC6蛋白水平的影响。沉默内源性P62和过表达外源性P62,可以分别抑制和促进自噬体降解。BPV(phen)导致HDAC6与P62相互作用减弱,释放HDAC6。BPV(phen)通过P62-HDAC6通路抑制自噬体降解。综上所述,本研究发现BPV(phen)抑制细胞增殖、促进细胞凋亡和细胞焦亡,同时抑制自噬体降解。其影响自噬体降解的分子通路可能是,BPV(phen)促进P62蛋白酶体降解,后者增强HDAC6活性,继而HDAC6对乙酰化微管蛋白的去乙酰化作用增加,影响自噬体的转运。
[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.

【学位授予单位】：安徽医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R96

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