探讨HSP90在主动脉夹层过程中对平滑肌表型转化的作用

发布时间：2018-03-11 21:50

本文选题：主动脉夹层　切入点：热休克蛋白90抑制剂　出处：《第二军医大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景及目的:热休克蛋白(HSPs)作为一种含量极其丰富同时在生物进化过程中高度保守的蛋白质家族,当细胞内环境紊乱或受到外界刺激时会显著上调以增强细胞应激能力及适应能力。热休克蛋白90(HSP 90)作为HSPs家族中的成员之一,在众多生理及病理过程中均起到重要的作用,近些年随着HSP 90抑制剂的研究逐步深入,目前以17-AAG为代表药物的格兰德霉素衍生物已经进入诸多血液病及实体瘤单药治疗II期临床实验。而HSP 90在心血管领域的作用机制也在逐步深入进行,目前已有研究表明,HSP90抑制剂17-AAG对于稳定动脉粥样硬化斑块有一定的作用,相关的研究也在肺动脉高压及腹主动脉瘤中看到了一定的成效,但其在主动脉夹层发生发展过程中的作用目前尚无报道。主动脉夹层(AD)最为危重的急症之一,尽管目前的诊疗水平已有了明显提升,但因夹层急性破裂而导致的院外死亡率仍居高不下,目前对于主动脉夹层的机制性研究认为,除了弹力纤维的退行性变外,血管平滑肌细胞(VSMCs)生物学特性的改变是参与大动脉疾病中的重要部分,因此,针对平滑肌细胞的机制研究也正在逐步成为了解大动脉疾病的突破点。本实验首先旨在明确HSP90在AD疾病发生发展中血清及组织的表达水平并推测其是否与平滑肌细胞表型转化存在一定的相关性,接下来在细胞水平明确HSP90抑制剂是否对VSMCs的表型转化具有影响,同时验证其对VSMCs生物学特性的影响,最后在AD的动物模型中应用HSP90抑制剂观察其对疾病的治疗效果。方法:收集20例从2014年9月至2015年3月在长海医院心血管外科因急性Stanford A型胸主动脉夹层行急诊手术患者的血清及术中胸主动脉组织,同期选取20例志愿者血清及8例心脏移植供体正常主动脉组织作为对照。液氮标本用于提取组织RNA行定量PCR、提取组织蛋白行Western blot;多聚甲醛固定后的标本行常规染色及免疫组化染色。胶原酶消化法获得SD大鼠原代胸主动脉平滑肌细胞,通过PDGF-bb诱导大鼠VSMCs发生生物学性状的改变,用HSP90抑制剂17-DMAG进行干预,对VSMCs的生物学功能进行检测,包括平滑肌细胞表型相关标志物RNA及蛋白水平的改变、CCK 8法测定增殖、平板划线法测定迁移、流式细胞仪检测细胞周期及凋亡。选取60只4周龄雄性FVB小鼠,通过β-BAPN喂养联合血管紧张素II建立主动脉夹层小鼠模型,并利用HSP90抑制剂17-DMAG干预模型,通过建模成功率及死亡率的比较以期获得HSP90抑制剂是否能防治主动脉夹层,并对不同组中主动脉组织提取组织蛋白行Western blot;多聚甲醛固定标本行常规HE染色及弹力纤维染色。结果:1、大体标本中,主动脉夹层患者主动脉组织中HSP90明显升高,且表达定为于血管壁中膜层平滑肌细胞,并且推测HSP90的表达升高与平滑肌细胞合成型标志物呈正相关;2、在细胞水平上,HSP90抑制剂17-DMAG能抑制由PDGF-bb诱导的平滑肌细胞的表型转化,同时能抑制由PDGF-bb诱导的平滑肌细胞增殖、迁移及周期改变。3、动物模型水平,HSP90抑制剂17-DMAG能显著降低胸主动脉夹层的死亡率,并且在组织学上明显缓解了动脉血管壁结构的破坏;同时在蛋白水平证明17-DMAG能缓解在主动脉夹层过程中平滑肌细胞的表型转化。结论:HSP90抑制剂17-DMAG能通过抑制平滑肌细胞表型转化减轻主动脉组织病变并减少AD模型的死亡率,从而可能成为防治主动脉夹层的潜在药物。
[Abstract]:Background and objective: heat shock protein (HSPs) as a kind of extremely rich content at the same time in the process of biological evolution of highly conserved protein family, when environmental disorder cells or by external stimulation will increase to enhance cellular stress ability and adaptability. Heat shock protein 90 (HSP 90) as a member of HSPs in the family, in many physiological and pathological processes play an important role in recent years, with the research of HSP 90 inhibitors gradually thorough, currently represented by 17-AAG drug geldanamycin derivatives have entered many hematological and solid tumor monotherapy phase II clinical trials. HSP 90 in the field of cardiovascular mechanism also in step by step, studies have shown that HSP90 inhibitor 17-AAG has a certain role in the stabilization of atherosclerotic plaques, related studies in pulmonary hypertension and abdominal aorta The aneurysm saw some success, but its role in the development of aortic dissection. There are no reports of aortic dissection (AD) is one of the most critical emergency, although the treatment level has been improved significantly, but the hospital mortality caused by acute rupture of dissection is still high, the current mechanism study for aortic dissection, except for degenerative change of elastic fibers, vascular smooth muscle cells (VSMCs) biological characteristics change is involved in the important part, large artery disease in the study for mechanism of smooth muscle cells is also gradually become the breakthrough point to understand large artery disease. This paper aims to make clear in HSP90 the expression level of serum and tissue AD in the occurrence and development of diseases and speculate whether the transformation is associated with smooth muscle cell phenotype in HSP cells, the next is water level 90 inhibitors of the VSMCs phenotype transformation has influence, and to verify the effect of the biological characteristics of VSMCs, the last in an animal model of AD in application of HSP90 inhibitors to observe the treatment effect of the disease. Methods: 20 cases were collected from September 2014 to March 2015 in Changhai Hospital of cardiovascular surgery for patients with acute Stanford type A aortic dissection for emergency operation of thoracic aorta in serum and tissue, selected 20 healthy volunteers and 8 patients with heart transplantation serum normal aortic tissues as the control group. Specimens were used to extract RNA liquid nitrogen quantitative PCR, extract tissue protein Western blot; paraformaldehyde fixed specimens after routine staining and immunohistochemical staining of aortic smooth muscle. SD cells in primary rat thoracic obtained by collagenase digestion, induced VSMCs in rats by PDGF-bb biological characteristics change with HSP90 inhibitor 17-DMAG Intervention on the biological functions of VSMCs were detected, including the phenotype of smooth muscle cell markers RNA and protein level changes, determination of the proliferation of CCK 8 determination method, transfer plate method, detection of cell cycle and apoptosis by flow cytometry. A total of 60 4 week old male FVB mice, by feeding combined with vascular beta -BAPN angiotensin II to establish mouse model of aortic dissection, and the use of HSP90 inhibitor 17-DMAG intervention model, by modeling the success rate and mortality in comparison to obtain whether HSP90 inhibitors can prevent aortic dissection, and the aortic tissue in different groups of Western extract tissue protein blot; paraformaldehyde fixed specimens stained with HE and elastic fiber staining results: 1 specimens, HSP90 aortic tissue of patients with aortic dissection were significantly increased, and the expression for the film in smooth muscle cells in the vascular wall, and that HSP90 The expression was positively correlated with the increased synthesis of smooth muscle cells; 2, at the cellular level, the transformation phenotype of HSP90 inhibitor 17-DMAG can inhibit PDGF-bb induced smooth muscle cells, and can inhibit the proliferation of smooth muscle cells induced by PDGF-bb, migration and cycle change of.3, animal model level, HSP90 inhibitor 17-DMAG can significantly reduce the thoracic aorta dissection and mortality, histologically relieved arterial wall damage; at the same time at the protein level that 17-DMAG can ease the transformation in the table type aortic dissection of smooth muscle cells. Conclusion: HSP90 inhibitor 17-DMAG can inhibit smooth muscle cell phenotype and reduce aortic tissue lesions AD model to reduce mortality, which may as a potential drug for prevention and treatment of aortic dissection.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.1

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