【作者】 许科帝；

【导师】 郑筱祥；

【作者基本信息】 浙江大学 ， 生物医学工程， 2010， 博士

【摘要】 动脉粥样硬化疾病是现代社会中最为常见的心血管疾病之一,其发病机理复杂,是一种多致病因素的高发病率疾病。在动脉粥样硬化的病理过程中,泡沫化细胞的出现和动脉粥样硬化斑块的形成被认为是动脉粥样硬化的病理特征。在病交后期,动脉粥样硬化斑块区域内过量的平滑肌细胞损伤及死亡直接影响了斑块区域结构的完整性,降低斑块稳定性,最终导致斑块破裂并形成血栓,造成血管堵塞,因此平滑肌细胞的死亡在动脉粥样硬化发病过程中起了重要作用。在体内,泡沫化细胞的来源主要是巨噬细胞和平滑肌细胞。在以往的实验中,泡沫化细胞的研究主要集中于巨噬细胞的泡沫化模型,对于平滑肌细胞来源的泡沫化细胞模型研究较少。最近的研究发现,细胞内过量的游离胆固醇可能是造成巨噬细胞源泡沫化细胞死亡的原因之一。游离胆固醇可能通过激活线粒体凋亡通路和内质网凋亡通路导致巨噬细胞源泡沫化细胞的凋亡。但细胞内过量游离胆固醇和平滑肌细胞死亡之间的联系还未见报道。本论文采用大鼠胸／腹主动脉平滑肌细胞和水溶性胆固醇(CHOL:MβCD)共孵育的方法建立起平滑肌来源的泡沫化细胞模型,并结合细胞内酰基辅酶A：胆固醇酰基转移酶(ACAT)的特异性阻断剂Sandoz58035,建立起游离胆固醇过载的平滑肌细胞模型。通过对病变平滑肌细胞内的钙离子、线粒体钙、活性氧(ROS)浓度、线粒体形态结构、线粒体膜电位等观察分析以及线粒体和内质网凋亡通路上相关蛋白的表达情况的测定对游离胆固醇造成的平滑肌细胞早期损伤和细胞凋亡坏死机制作出了初步的研究。本论文还通过观察自噬小体的形成以及自噬特异性标志蛋白LC3含量变化的测定检测了平滑肌细胞在游离胆固醇刺激下自噬的激活情况。结合药物干扰自噬在平滑肌细胞内的激活,从细胞和分子水平研究了游离胆固醇激活平滑肌细胞内自噬产生的发生机制。本论文对游离胆固醇过载情况下平滑肌细胞内一系列生理病理的变化进行了模拟,对相关细胞机制进行了分析,为深入理解动脉粥样硬化的发病机理和发病过程提供了理论基础。本论文得到的主要研究成果有：1.利用平滑肌细胞和水溶性胆固醇共孵育建立起平滑肌细胞源的泡沫化细胞模型。同时利用ACAT酶特异性阻断剂Sandoz58035作用于平滑肌源的泡沫化模型建立游离胆固醇过载的细胞模型,模拟了动脉粥样硬化中后期平滑肌细胞的病变过程。2.利用泡沫化模型和游离胆固醇过载模型,比较两类细胞模型内钙离子,线粒体钙离子以及细胞内ROS含量水平的变化。实验发现在游离胆固醇载入过程中细胞内钙离子和线粒体钙离子浓度出现明显下降,而ROS含量随孵育时间的增加而上升。在正常细胞和泡沫化平滑肌细胞中这三种物质浓度基本维持在稳定的水平,验证了细胞内多余的游离胆固醇影响了平滑肌细胞内环境的稳态。3.对平滑肌细胞的线粒体进行荧光标记和线粒体形态结构动力学的实时观察、记录、分析。发现在游离胆固醇过载的平滑肌细胞中,线粒体在游离胆固醇载入后4-8小时内发生断裂,由正常状态下的网状转变为碎点状。4.从线粒体介导的细胞凋亡通路以及内质网介导的细胞凋亡通路对游离胆固醇造成的平滑肌细胞凋亡坏死进行进一步的分析。实验发现游离胆固醇的载入可以导致线粒体膜电位的崩解,改变细胞内bcl-2、bax蛋白含量并导致线粒体内cyto c的释放。同时内质网压力蛋白KDEL表达量在游离胆固醇的刺激下大大上升并激活UPR反应过程中CHOP蛋白的表达。说明游离胆固醇通过线粒体凋亡通路和内质网凋亡通路的激活造成细胞死亡。5.对游离胆固醇过载的平滑肌细胞内自噬的水平进行了研究。发现游离胆固醇的载入可以导致平滑肌细胞内发生自噬。同时发现自噬的程度和游离胆固醇载入的浓度和时间均有关系。利用自噬的阻断剂3-MA和增强剂rapamycin说明自噬的产生减轻了游离胆固醇造成的平滑肌细胞损伤。进一步的实验还发现自噬通过清除损伤的细胞器如线粒体来实现对游离胆固醇造成的平滑肌损伤的保护作用,但同时过量的自噬也有可能直接导致平滑肌细胞出现自噬样坏死。细胞内过量的游离胆固醇对线粒体、内质网等细胞器的功能结构造成损伤。轻度损伤的细胞器可以通过激活自噬的发生得以清除,从而减轻细胞器损伤带来的细胞压力。过量的细胞器损伤则可以激活细胞内凋亡信号通路,造成平滑肌细胞的死亡。同时细胞内过量的自噬也可能直接造成平滑肌出现自噬样死亡。因此,游离胆固醇造成的平滑肌细胞损伤是一个由细胞凋亡,自噬和细胞坏死等多种机制调控的复杂细胞死亡事件。本论文的创新点主要有以下几点：1.利用水溶性胆固醇和ACAT酶的抑制剂和平滑肌细胞共孵育来建立泡沫化和游离胆固醇过载的平滑肌细胞模型在国内研究中还较少见,很好的解决了平滑肌来源的泡沫化细胞模型稳定性、重复性差的问题。两类细胞模型分别模拟了动脉粥样硬化发病过程中平滑肌细胞可能出现的病理变化,实现了体外建立起平滑肌细胞泡沫化病变的病理模型。2.本研究从线粒体形态结构的动力学变化着手,结合线粒体凋亡通路上多个作用靶点的深入研究,提出线粒体是游离胆固醇造成的平滑肌损伤的重要作用靶点。同时通过对内质网结构功能的研究也验证了游离胆固醇可以直接作用于内质网上并和线粒体通路一样对平滑肌细胞的功能状态有着直接的影响,并且这两条信号通路相互之间是协同作用的。通过多条细胞内信号通路的研究比较完整的解释游离胆固醇造成平滑肌细胞损伤的细胞机制。3.本研究利用多种技术手段验证了自噬在游离胆固醇过载的平滑肌细胞中的激活。结合自噬特异性的抑制剂和激动剂等方法,对自噬在游离胆固醇造成的平滑肌损伤过程中可能的细胞机制做出了解释。通过实验验证了自噬在游离胆固醇造成的平滑肌细胞损伤过程中起到了一定的保护作用,并且这一作用是通过保护线粒体、内质网等细胞器的功能来实现的。综合上述的各个方面,本研究通过建立起稳定的平滑肌细胞源泡沫化细胞模型,从细胞凋亡和自噬发生两方面对游离胆固醇造成的平滑肌细胞损伤的细胞机制作出了完整的解释,为预防动脉粥样硬化疾病和药物研究提供相应的理论基础。更多还原

【Abstract】 Atherosclerosis is one of the most common cardiovascular diseases in the modern society. Several important environmental and genetic risk factors have been associated with atherosclerosis, including hypertension, high blood cholesterol, diabetes, obesity, gender and unhealthy lifestyles. The appearance of cholesterol and cholesteryl esters-laden foam cells in advanced atherosclerotic lesions is a hallmark of atherosclerosis. In the advanced plaques, excessive death of smooth muscle cells (SMCs) may compromise plaque integrity, weaken the stability of fibrous cap, lead to plaque rupture and finally trigger thrombosis and vessel occlusion.The original of foam cell was identified both from macrophages and SMCs. Most research works of the foam cell was focused on macrophage-original cell models. Recent studies have found that excess intracellular free cholesterol (FC) is the reason of macrophage-derived foam cell death. FC-overloading activated both endoplasmic reticulum-(ER) and mitochondrial-dependent apoptosis pathway and induced macrophage apoptosis. However, little is known about the relationship between excessive cytoplasmic cholesterol and SMCs death.In present study, rat smooth muscle cells were loaded with cholesterol:methyl-β-cyclodextrin (CHOL:MPCD) complexes accompanied with the ACAT inhibitor Sandoz58035. The potential impairment of the cellular calcium concentration, mitochondrial calcium concentration, ROS concentration and the dynamic change of the mitochondrial morphology and mitochondrial membrane potential were analyzed. We also estimated the cytotoxicity of excess FC and detected the activation of related mitochondrial-and ER-dependent cell apoptosis proteins. In order to evaluate the activation of autophagy in FC-overloading SMCs, we observed the conversion of microtubule-associated protein-1 light chain 3-I (LC3-I) to a phosphatidylethanolamine-conjugated form (LC3-II) and the formation of autophagic vacuoles (AVs). Combined with pharmacology experiments, we further explained the cellular and molecular mechanism of the activation of autophagy in SMCs. In conclusion, our experiments simulated the physiological and pathological changes of the SMCs under the FC-overloading condition and demonstrated that FC-overloading SMCs underwent a complex mode of cell death, including cell apoptosis and autophagy.The major results of our study were:1. Using CHOL:MβCD to establish the SMC-derived foam cell model. The FC-overloading cell model was then established with CHOL:MPCD accompanied with the ACAT inhibitor Sandoz58035.2. Compared with foam cell model, we found that the intracellular calcium concentration and mitochondrial calcium concentration were down-regulated while the ROS concentration was up-regulated in the FC-overloading cell model.3. After incubated with CHOL.MβCD plus 58035 for 4-8 h, the normal tubular mitochondrial network was completely converted to a punctual conformation.4. Our experiments herein revealed a decrease of mitochondrial membrane potential in FC-overloading SMCs. After a widespread mitochondrial dysfunction, FC-overloading caused an increase of cellular Bax accompanied with a decrease of Bcl-2 and finally caused the release of cytochrome C. Our experiments also showed that treatment with excess FC induced ER stress and caused UPR-mediated SMCs death, as demonstrated by upregulation of KDEL and inducing of CHOP, respectively.5. The excess intracellular FC led to a large-scale cellular organelles damage which further activated formation of AVs and LC3 processing. Enhanced autophagy played as a prosurvival mechanism that prevented cell death in FC-overloading SMCs.In this paper, the usage of water-soluble cholesterol and ACAT inhibitor to create SMC-original foam cell model and FC-overloading cell model were more stable and repeatable. Our experiments explained the cellular mechanism of FC-overloading induced SMCs damage through multiple intracellular signaling pathways. And the concept of autophagy was introduced into these cell models for the first time which is also an innovative point in this study.更多还原