自噬在血管损伤中的作用和分子机制

发布时间：2018-06-25 22:16

  本文选题：血管内皮细胞 + 线粒体自噬　； 参考：《山东大学》2016年博士论文

【摘要】：研究背景自噬是细胞将自身受损的细胞器或错误折叠的蛋白质包裹、吞噬,并运输到溶酶体,降解,实现自身物质代谢更新,能量再循环利用的生物学过程。自噬对维持血管内环境的稳态的十分重要,并且参与了血管内各种细胞生长和死亡的调控。在血管损伤动物模型中,伴随着血管内皮细胞的首先死亡和血管平滑肌细胞的不断增殖,这两种细胞中都存在着不同程度的自噬反应。血管内皮细胞、血管平滑肌细胞是构成血管组织最主要的两种细胞类型,深入探讨自噬对这两种血管细胞的作用及调节机制意义重大。首先,血管内皮细胞活跃地参与血管各功能活动,因此内皮细胞能量代谢要迅速且准确,而细胞内的能量供应站即线粒体,这个细胞器自身的稳态和代谢更新也就更为重要。研究表明,在细胞中,通过自噬机制可以选择性地清除那些受到损伤或者已经老化的线粒体,也就是线粒体自噬,来维护线粒体结构与功能的完整性。在代谢综合征中,内皮细胞中出现线粒体损伤、积聚,细胞内ROS水平升高,而这些代谢应激诱导的损伤线粒体又进一步损伤了内皮细胞的功能,加重了心血管疾病。PINK1/Parkin这条信号通路在控制线粒体自噬的过程中发挥了重要的作用。当PINK1/Parkin分子突变后会引起神经系统的帕金森疾病。因此研究PINK1/Parkin信号通路介导的线粒体自噬在代谢应激中对内皮细胞的作用机制,愈来愈重要。平滑肌细胞是血管中除内皮细胞外另一重要细胞群,在血管损伤后的血管修复与重构中,平滑肌细胞的增殖成为主要的细胞生命活动,因此研究自噬对血管损伤后平滑肌细胞的调控,也就显得十分重要。但是近年来无论是高血压,还是其它代谢性疾病等引起的血管疾病中,仅仅采用针对平滑肌细胞的药物并没有能够有效的控制血管疾病病情的发展,更不用说根治血管疾病。例如在严重的冠脉阻塞中,冠状动脉旁路移植术仍然是最有效的外科治疗手段,但是,术后十年,50%的移植血管会发生再阻塞,这主要归因于血管平滑肌细胞的不断增殖和血管新生内膜的持续增生。接着人们采用直接或是间接的方法抑制血管平滑肌细胞的增殖,但是20年来,冠状动脉旁路移植术术后十年再阻塞的概率并没有得到很好的改善。因此针对抑制平滑肌细胞的治疗就显得让人迟疑,但同时这也引发了人们新的思考,是否我们忽略了更为重要的治疗耙点?已有证据显示血管中存在着大量的血管干细胞,血管损伤后,这些干细胞会被激活。在小鼠静脉移植这个模型中,徐清波教授已经证明移植静脉新生内膜中,增殖的平滑肌细胞60%来源于供体静脉血管。在我们的实验中发现,移植术后3天,移植静脉血管原有的成熟平滑肌细胞都已死亡,而血管外膜中Sca-1(干细胞抗原-1)阳性的干细胞仍存活,并且在术后7-14天增殖达到高峰,这群干细胞无疑是构成移植静脉血管新生内膜的主要来源。干细胞是一种长寿命细胞,长期处于静息状态。这种状态的维持,需要严密的自我复制和分化潜能。自噬作为一种可诱导的分解代谢的细胞生命活动,对于细胞内蛋白质质量控制及细胞器物质代谢再利用起着重要作用。按照发育阶段分类干细胞为：胚胎干细胞和成体干细胞。自噬在这些干细胞中的功能及作用已被不同的文献报道：比如在胚胎形成过程中,自噬可以帮助清除死亡细胞,移除有缺陷的线粒体及某些泛素化的蛋白质,维持胚胎干细胞的增殖,降解在其分化过程中形成的中间体。在造血干细胞中作用,自噬主要表现为维持稳定的增殖,有效移除有缺陷的线粒体,阻止DNA损伤,自我复制。自噬在间充质干细胞中的作用则主要是细胞保护反应和促进分化。自噬在神经干细胞中的作用则是促进分化及营养不足时,维持存活。总之自噬对干细胞的生长和分化起到了严密的质量监控,正因为自噬功能的存在,各种干细胞才能一方面维护自体干性状态,另一方面分化成成熟的不同的体细胞完成机体的各种修复过程。目前对血管干细胞和自噬相关的研究较少,但这无疑为治疗血管损伤性疾病提供了新的方向。我们的研究则聚焦在寻找可以调控自噬的去泛素化酶分子CYLD(圆柱瘤基因编码的蛋白)上面,CYLD可以通过激活mTOR信号通路,来控制自噬,进而干预血管干细胞的激活、自我复制及分化潜能,维持血管内环境稳态,达到治疗血管病的最终目的。研究目的第一部分研究线粒体自噬对游离脂肪酸代谢应激诱导的内皮细胞损伤的保护机制：1研究线粒体自噬对维持内皮细胞线粒体结构和功能完整性的作用。2研究PINK1/Parkin介导的线粒体自噬对游离脂肪酸(FFA)诱导的内皮细胞损伤的保护机制。第二部分研究CYLD是否通过控制自噬,调节血管干细胞的功能,进一步影响血管损伤后的重构过程：1在同种异体小鼠颈外静脉向颈总动脉移植模型中,解析血管干细胞在移植静脉新生内膜形成过程中的巨大贡献。2在CYLD基因敲除小鼠颈外静脉向野生型小鼠颈总动脉移植模型中,分析移植静脉新生内膜形成情况。3 CYLD是否可以调控自噬,干预血管干细胞的自我更新和向前体细胞分化功能,最终影响血管重构。研究方法第一部分1动物模型1.1Ⅰ型糖尿病小鼠模型：C57BL/6J小鼠,50 mg/kg STZ连续注射五天,10天后检测小鼠血糖水平、体重及4周后进行胸主动脉的取材固定。1.2高脂喂养小鼠模型：用高脂饲料对C57BL/6J小鼠进行持续喂养,三个月后检测小鼠的血脂水平及进行胸主动脉的取材固定。2细胞培养和转染：2.1用含有5%的胎牛血清(FBS)、内皮细胞生长因子、双抗等物质的内皮细胞培养基培养人主动脉弓内皮细胞(HAECs)。2.2用lipofectamine 2000对PINK1/Parkin的小干扰RNA及过表达质粒进行内皮细胞的转染。3棕榈酸(PA)的配制：将PA溶入200 mmol/L的甲醇后,加入10%牛血清白蛋白(BSA), PH=7.5,-20℃冻存。4 Western blot实验分析：使用Western细胞裂解液裂解不同刺激条件处理的细胞,检测蛋白浓度,使用10% SDS-PAGE进行电泳,免疫印迹法检测不同目的蛋白的表达情况。5细胞免疫荧光染色：将不同刺激条件处理后的细胞接种在细胞爬片上面,固定后,一抗二抗孵育,封片,用Ultra VIEW VOX共聚焦显微镜观察,拍照,最后用Volocigy软件分析输出高质量图片。6线粒体检测：使用Mito Tracker Deep Red染色试剂盒进行内皮细胞线粒体染色。7线粒体自噬检测：使用Mito Tracker Deep Red进行内皮细胞线粒体染色的同时,进行自噬小体标志物LC3或溶酶体标志物Lamp1的共定位染色。8透射电镜检测：将不同刺激条件处理后的细胞用戊二醛固定过夜,第二天送往济南微亚生物技术公司,由该公司进行透射电镜的检测JEOL-1200EX透射电镜观察,MORADA-G2记录结果。9线粒体膜电位的检测：羰基氰-3-氯苯腙(CCCP)是一种强效的线粒体氧化磷酸化解偶联剂,用它或PA处理内皮细胞后进行四甲基罗丹明甲酯(TMRM)和Mito Tracker Deep Red线粒体共定位双染色,用Ultra VIEW VOX共聚焦显微镜进行活细胞观察,拍照,最后用Volocigy软件分析输出高质量图片。10 ROS水平的检测：将不同刺激条件处理后的内皮细胞用ROS检测试剂盒进行检测。11 ATP水平的检测：将不同刺激条件处理后的内皮细胞用ATP检测试剂盒进行检测。12原位末端标记法(TUNEL):使用罗氏TUNEL检测试剂盒检测不同刺激条件处理后的内皮细胞凋亡情况。13数据分析：使用Student's t检验或单因素方差分析进行统计学分析。当P0.05认为有统计学差异意义。第二部分1静脉移植模型：用带有柄和体的CUFF管进行颈外静脉的移植,移植后,观察静脉血管中有动脉血液流通,及血管搏动,说明手术成功。2用药：2.1局部用药：辛伐他汀(30umol/L)/SenexinA (3 umol/L)用20% F-127胶包裹在移植血管周围。2.2全身系统用药：辛伐他汀(1.6mg/kg)/SenexinB (20 mg/kg)分别采用灌胃和腹腔内注射的方法。用药间为：术前三天至术后28天连续用药或术后三天至术后28天连续用药。3组织免疫荧光染色法：分别在术后0、1、3、7、14、21、42天后取材,固定,近心端开始切片,切片厚度为5μm,连续切片,每隔501μm或是500μm各取一张切片。进行HE染色和组织免疫荧光染色。4细胞培养：取8周大C57BL/6J小鼠一只,取出胸主动脉及根部,显微镜下分离出外膜,剪碎,并用胶原酶Ⅱ进行适度消化后,过滤血管碎片,用干细胞培养基培养数天,当细胞长到足够数量后,用Sca-1磁珠钓取Sca-1阳性细胞,再进行培养。5统计分析：使用Student's t检验或单因素方差分析进行统计学分析。当P0.05认为有统计学差异意义。研究结果第一部分1 PA处理后内皮细胞自噬和线粒体自噬均增强。2 PINK1/Parkin信号通路参与了PA诱导内皮细胞的线粒体自噬：3 PINK1/Parkin信号通路保护了PA诱导的内皮细胞中损伤的线粒体的膜电位：4 PINK1/Parkin信号通路阻止了PA诱导内皮细胞中线粒体失功能。5 PINK1/Parkin信号通路阻止了PA诱导内皮细胞死亡。6在肥胖小鼠和Ⅰ型糖尿病小鼠模型中,PINK1/Parkin信号通路在小鼠血管中被激活。第二部分：1在同种异体小鼠颈外静脉向颈总动脉移植模型中,抑制血管干细胞后,移植静脉的新生内膜也得到抑制。1.1在静脉移植早期,供体血管平滑肌细胞死亡,同时伴有血管干细胞的增殖,分化最终参于移植静脉新生内膜的形成。1.2建立了一个更全面的可以定量测量和分析移植静脉新生内膜中平滑肌细胞的方法。1.3在移植术后前三天,成熟平滑肌细胞死亡而血管干细胞存活并增殖最终参于血管新生内膜的形成。1.4辛伐他汀可能主要通过抑制早期血管干细胞的激活来抑制移植术后血管新生内膜的形成与增生。1.5 Senexin A/B, CDK8抑制物,可以通过抑制移植术后血管干细胞的激活而非抑制血管平滑肌细胞来抑制血管新生内膜的形成与增生。2在CYLD基因敲除小鼠颈外静脉向野生型小鼠颈总动脉移植模型中,CYLD基因的敲除促进了血管新生内膜形成。3 CYLD通过激活mTOR信号通路,抑制自噬。研究结论1 PINK1/Parkin信号通路通过线粒体自噬对内皮细胞维持线粒体功能完整性发挥了重要作用,可以清除代谢应激中受损线粒体,从而保护了内皮细胞的功能。2血管干细胞参于血管损伤后新生内膜形成过程并发挥了重要作用,CYLD通过调控mTOR信号通路来调控自噬,进而干预血管干细胞的自我更新,向前体细胞的分化,增殖,最终影响血管重构。
[Abstract]:Background autophagy is an autophagy that is encapsulated by a cell or a wrongly folded protein, phagocytic, and transported to the lysosome, degrading, realizing the metabolic renewal of its own material, and the biological process of energy recirculation. Autophagy is important to maintain the homeostasis of the intravascular environment and participates in the growth and death of various cells in the blood vessels. In the animal model of vascular injury, with the first death of vascular endothelial cells and the continuous proliferation of vascular smooth muscle cells, there are different degrees of autophagy in these two cells. Vascular endothelial cells, vascular smooth muscle cells are the two most important types of vascular tissue, and the autophagy is discussed in depth. The role and regulation mechanism of these two kinds of vascular cells are of great significance. First, vascular endothelial cells actively participate in various functional activities of blood vessels, so the energy metabolism of endothelial cells should be quickly and accurately, and the energy supply station in the cell is mitochondria, and the homeostasis and metabolic renewal of this organelle itself is more important. In the metabolic syndrome, mitochondrial damage, accumulation, elevated levels of ROS in cells, and these metabolic stress induced damage lines, are possible to maintain mitochondrial structure and function integrity in the metabolic syndrome. The body further damages the function of endothelial cells and aggravates the cardiovascular disease.PINK1/Parkin signaling pathway plays an important role in controlling mitochondrial autophagy. When the PINK1/Parkin molecule mutates, it will cause the Parkinson disease of the nervous system. Therefore, the study of the mitochondrial autophagy mediated by the PINK1/ Parkin signaling pathway Smooth muscle cells are another important cell group except endothelial cells in the blood vessels. The proliferation of smooth muscle cells is the main cell life activity in vascular repair and reconstruction after vascular injury. Therefore, the regulation of self macrophages on vascular smooth muscle cells after vascular injury is also studied. It is important, but in recent years, in vascular diseases caused by hypertension and other metabolic diseases, only the use of drugs aimed at smooth muscle cells has not been able to effectively control the development of vascular disease, let alone to cure vascular diseases. For example, coronary artery bypass graft in severe coronary occlusion. Surgery is still the most effective surgical treatment. However, ten years after the operation, 50% of the transplanted vessels will be re blocked, which is attributed to the continuous proliferation of vascular smooth muscle cells and the continuous proliferation of neointima in the vascularization. Then, the proliferation of vascular smooth muscle cells is suppressed by direct or indirect methods, but for 20 years, coronary movement has been performed. The probability of re blocking ten years after the vein bypass grafting has not been well improved. Therefore, the treatment of the suppression of smooth muscle cells appears to be hesitant, but it also causes new thinking. Do we ignore the more important treatment rake? There is evidence that there are a large number of vascular stem cells in the blood vessels, blood. In the mouse vein transplant model, Professor Xu Qingbo has proved that 60% of the proliferating smooth muscle cells are derived from the donor vein. In our experiment, we found that the original smooth muscle cells of the vein graft were dead at 3 days after the transplantation. The stem cells of the Sca-1 (stem cell antigen -1) positive in the epicardium still survive and proliferate at the peak 7-14 days after the operation. This group of stem cells is undoubtedly the main source of the neointima of the vein graft. The stem cells are a long life life cell and remain resting for a long time. Autophagy. Autophagy, as an induced metabolic cell life activity, plays an important role in intracellular protein quality control and organelle metabolism reutilization. Stem cells are classified as stem cells and adult stem cells according to the developmental stages. The function and function of autophagy in these stem cells has been different In the process of embryogenesis, autophagy, for example, helps to remove dead cells, remove defective mitochondria and some ubiquitin proteins, maintain the proliferation of embryonic stem cells, and degrade intermediates formed during their differentiation. In hematopoietic stem cells, autophagy is mainly shown to maintain stable proliferation and be effectively removed. The function of autophagy in mesenchymal stem cells is mainly cell protection and promotion of differentiation. Autophagy plays a role in promoting differentiation and insufficient nutrition in neural stem cells. In short, autophagy is closely monitored for the growth and differentiation of stem cells. Due to the existence of autophagy, all kinds of stem cells can maintain autologous dry state on the one hand and differentiate into mature different somatic cells to complete various repair processes. There are few studies on vascular stem cells and autophagy, but this will undoubtedly provide a new direction for the treatment of vascular injury. It is focused on the search for autophagy, which regulates the autophagy molecule CYLD (the protein encoded by the cylindric gene). CYLD can control autophagy by activating the mTOR signaling pathway, and then interferes with the activation of vascular stem cells, self replication and differentiation potential, and maintains the homeostasis of vascular intravascular environment to achieve the ultimate goal of treating vascular diseases. The first part study the protective mechanism of mitochondrial autophagy induced endothelial cell damage induced by free fatty acid metabolism stress: 1 study the role of mitochondrial autophagy to maintain mitochondrial structure and functional integrity of endothelial cells.2 study the protection of mitochondrial autophagy mediated endothelial cell injury induced by free fatty acid (FFA) induced by PINK1/Parkin Mechanism. The second part studies whether CYLD regulates the function of vascular stem cells by controlling autophagy and regulates the function of vascular stem cells and further affects the reconstruction process after vascular injury. 1 in the allograft mouse model of external jugular vein to common carotid artery transplantation, the great contribution of.2 to CYLD gene knockout in the neointima formation process of the transplanted vein In the model of common carotid artery transplantation in mice from the external jugular vein to the wild type mouse, the analysis of the formation of neointima of the transplanted vein.3 CYLD can regulate autophagy, interfere with the self renewal of the vascular stem cells and the function of the differentiation of the forward body cells, and ultimately affect the vascular remodeling. The first part of the 1 animal model of the animal model of type 1.1 diabetic mice C57BL/6J mice, 50 mg/kg STZ continuous injection for five days, 10 days after the test of blood glucose level, body weight and 4 weeks after the thoracic aorta were used to fix the.1.2 high fat feeding mice model: high fat feed for continuous feeding of C57BL/6J mice, three months after the detection of blood lipid level in mice and the thoracic aorta material fixed.2 cell culture. Cultured and transfected: 2.1 the human aortic arch endothelial cells (HAECs).2.2 with 5% fetal bovine serum (FBS), endothelial cell growth factor, double resistance and other substances,.2.2 with Lipofectamine 2000 on PINK1/Parkin small interference RNA and overexpressed plasmids for.3 palmitic acid (PA) preparation: PA into 200 MMO After l/L methanol, adding 10% bovine serum albumin (BSA), PH=7.5, -20 C,.4 Western blot experimental analysis: using Western cell lysate to crack the cells treated with different stimulation conditions, detecting protein concentration, using 10% SDS-PAGE for electrophoresis, and detecting the expression of different target proteins by Western blot,.5 cell immunofluorescence staining After the cells treated with different stimulation conditions were inoculated on the cell climbing tablets, after immobilization, a anti two anti incubation and sealing film was observed and photographed with Ultra VIEW VOX confocal microscope. Finally, the Volocigy software was used to analyze the high quality images of.6 mitochondria: Mito Tracker Deep Red staining kit was used to carry out mitochondrial infection of endothelial cells. Autophagy detection of mitochondrial.7 mitochondria: using Mito Tracker Deep Red for mitochondrial staining of endothelial cells, simultaneous localization of autophagic marker LC3 or lysosome marker Lamp1 is carried out by transmission electron microscopy (TEM) detection of.8: the cells treated with different stimuli were fixed with glutaraldehyde for the night and sent to Ji'nan micro biotechnology for second days. The company, the company carries out transmission electron microscope examination JEOL-1200EX transmission electron microscope observation, MORADA-G2 record results.9 mitochondrial membrane potential detection: carbonyl cyanogen -3- chlorobenzene hydrazone (CCCP) is a powerful mitochondrial oxidative phosphorylation uncoupling agent, using it or PA treatment of endothelial cells after four methyl Luo Danming methyl Luo Danming methyl ester (TMRM) and Mito Tracker Deep Red mitochondrial co localization double staining, using Ultra VIEW VOX confocal microscope for living cell observation, photographing, and finally using Volocigy software to analyze the level of.10 ROS output of high quality pictures: the detection of.11 ATP level by ROS detection kit after different stimulation conditions: the different stimulus conditions will be detected. ATP detection kit was used to detect.12 in situ terminal labeling (TUNEL):.13 data analysis was used to detect the apoptosis of endothelial cells treated with different stimuli using Roche TUNEL Kit: Student's t test or single factor analysis of variance analysis. When P0.05 believed there was a statistical difference. Second part 1 vein transplantation model: transplantation of the external jugular vein with the CUFF tube with a handle and body. After transplantation, we observe the circulation of blood in the veins of the veins in the veins of the veins and the pulsation of the blood vessels. It shows that the operation is successful for.2 medication: 2.1 local medication: simvastatin (30umol/L) / SenexinA (3 umol/L) wrapped around the transplanted vascular.2 with 20% F-127 glue .2 systemic medication: simvastatin (1.6mg/kg) /SenexinB (20 mg/kg) were treated with intraperitoneal injection and intraperitoneal injection respectively. The use of the medication was continuous medication from three days before operation to 28 days after operation or three days after operation to 28 days after operation:.3 tissue immunofluorescence staining, respectively, after 0,1,3,7,14,21,42 after operation, fixed, near the end of the heart. The slice thickness was 5 mu m, sliced continuously, each 501 m or 500 micron m were sectioned. HE staining and tissue immunofluorescence staining.4 cell culture were taken to remove the thoracic aorta and the root of a large C57BL/6J mouse, the outer membrane was removed under microscope, and the collagenase II was used to digest the blood vessel fragments. After a few days in the stem cell culture medium, when the cells grew enough, the Sca-1 positive cells were caught with Sca-1 magnetic beads and then cultured for.5 statistical analysis: Student's t test or single factor analysis of variance analysis. When P0.05 believed that there was statistical difference. The results of the first part of the study were 1 PA after the endothelium autophagy. And mitochondrial autophagy enhanced the.2 PINK1/Parkin signaling pathway involved in the mitochondrial autophagy of PA induced endothelial cells: the 3 PINK1/Parkin signaling pathway protects the mitochondrial membrane potential damaged by PA induced endothelial cells: the 4 PINK1/Parkin signaling pathway prevents PA induced mitochondrial dysfunction.5 PINK1/Parkin signaling in the endothelial cells. The road prevented PA induced endothelial cell death.6 in the obese mice and the model of type I diabetic mice, the PINK1/Parkin signaling pathway was activated in the mouse blood vessels. Second: 1 in the allograft mouse model of the external jugular vein to the common carotid artery, the neointima of the vein graft is also suppressed after the suppression of the vascular stem cells. The vascular smooth muscle cells died at the early stage of vein transplantation, accompanied by the proliferation of vascular stem cells, and differentiation finally involved the formation of the neointima of the transplanted vein..1.2 established a more comprehensive method for quantitative measurement and analysis of the smooth muscle cells in the neointima of the transplanted vein..1.3 was mature smooth muscle cells three days after the transplantation. Death and vascular stem cells survive and proliferate and eventually participate in neointimal formation..1.4 simvastatin may mainly inhibit early vascular stem.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R363
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本文编号：2067729