發(fā)布時(shí)間：2018-03-11 21:50

  本文選題：主動(dòng)脈夾層　切入點(diǎn)：熱休克蛋白90抑制劑　出處：《第二軍醫(yī)大學(xué)》2017年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：研究背景及目的:熱休克蛋白(HSPs)作為一種含量極其豐富同時(shí)在生物進(jìn)化過(guò)程中高度保守的蛋白質(zhì)家族,當(dāng)細(xì)胞內(nèi)環(huán)境紊亂或受到外界刺激時(shí)會(huì)顯著上調(diào)以增強(qiáng)細(xì)胞應(yīng)激能力及適應(yīng)能力。熱休克蛋白90(HSP 90)作為HSPs家族中的成員之一,在眾多生理及病理過(guò)程中均起到重要的作用,近些年隨著HSP 90抑制劑的研究逐步深入,目前以17-AAG為代表藥物的格蘭德霉素衍生物已經(jīng)進(jìn)入諸多血液病及實(shí)體瘤單藥治療II期臨床實(shí)驗(yàn)。而HSP 90在心血管領(lǐng)域的作用機(jī)制也在逐步深入進(jìn)行,目前已有研究表明,HSP90抑制劑17-AAG對(duì)于穩(wěn)定動(dòng)脈粥樣硬化斑塊有一定的作用,相關(guān)的研究也在肺動(dòng)脈高壓及腹主動(dòng)脈瘤中看到了一定的成效,但其在主動(dòng)脈夾層發(fā)生發(fā)展過(guò)程中的作用目前尚無(wú)報(bào)道。主動(dòng)脈夾層(AD)最為危重的急癥之一,盡管目前的診療水平已有了明顯提升,但因夾層急性破裂而導(dǎo)致的院外死亡率仍居高不下,目前對(duì)于主動(dòng)脈夾層的機(jī)制性研究認(rèn)為,除了彈力纖維的退行性變外,血管平滑肌細(xì)胞(VSMCs)生物學(xué)特性的改變是參與大動(dòng)脈疾病中的重要部分,因此,針對(duì)平滑肌細(xì)胞的機(jī)制研究也正在逐步成為了解大動(dòng)脈疾病的突破點(diǎn)。本實(shí)驗(yàn)首先旨在明確HSP90在AD疾病發(fā)生發(fā)展中血清及組織的表達(dá)水平并推測(cè)其是否與平滑肌細(xì)胞表型轉(zhuǎn)化存在一定的相關(guān)性,接下來(lái)在細(xì)胞水平明確HSP90抑制劑是否對(duì)VSMCs的表型轉(zhuǎn)化具有影響,同時(shí)驗(yàn)證其對(duì)VSMCs生物學(xué)特性的影響,最后在AD的動(dòng)物模型中應(yīng)用HSP90抑制劑觀察其對(duì)疾病的治療效果。方法:收集20例從2014年9月至2015年3月在長(zhǎng)海醫(yī)院心血管外科因急性Stanford A型胸主動(dòng)脈夾層行急診手術(shù)患者的血清及術(shù)中胸主動(dòng)脈組織,同期選取20例志愿者血清及8例心臟移植供體正常主動(dòng)脈組織作為對(duì)照。液氮標(biāo)本用于提取組織RNA行定量PCR、提取組織蛋白行Western blot;多聚甲醛固定后的標(biāo)本行常規(guī)染色及免疫組化染色。膠原酶消化法獲得SD大鼠原代胸主動(dòng)脈平滑肌細(xì)胞,通過(guò)PDGF-bb誘導(dǎo)大鼠VSMCs發(fā)生生物學(xué)性狀的改變,用HSP90抑制劑17-DMAG進(jìn)行干預(yù),對(duì)VSMCs的生物學(xué)功能進(jìn)行檢測(cè),包括平滑肌細(xì)胞表型相關(guān)標(biāo)志物RNA及蛋白水平的改變、CCK 8法測(cè)定增殖、平板劃線法測(cè)定遷移、流式細(xì)胞儀檢測(cè)細(xì)胞周期及凋亡。選取60只4周齡雄性FVB小鼠,通過(guò)β-BAPN喂養(yǎng)聯(lián)合血管緊張素II建立主動(dòng)脈夾層小鼠模型,并利用HSP90抑制劑17-DMAG干預(yù)模型,通過(guò)建模成功率及死亡率的比較以期獲得HSP90抑制劑是否能防治主動(dòng)脈夾層,并對(duì)不同組中主動(dòng)脈組織提取組織蛋白行Western blot;多聚甲醛固定標(biāo)本行常規(guī)HE染色及彈力纖維染色。結(jié)果:1、大體標(biāo)本中,主動(dòng)脈夾層患者主動(dòng)脈組織中HSP90明顯升高,且表達(dá)定為于血管壁中膜層平滑肌細(xì)胞,并且推測(cè)HSP90的表達(dá)升高與平滑肌細(xì)胞合成型標(biāo)志物呈正相關(guān);2、在細(xì)胞水平上,HSP90抑制劑17-DMAG能抑制由PDGF-bb誘導(dǎo)的平滑肌細(xì)胞的表型轉(zhuǎn)化,同時(shí)能抑制由PDGF-bb誘導(dǎo)的平滑肌細(xì)胞增殖、遷移及周期改變。3、動(dòng)物模型水平,HSP90抑制劑17-DMAG能顯著降低胸主動(dòng)脈夾層的死亡率,并且在組織學(xué)上明顯緩解了動(dòng)脈血管壁結(jié)構(gòu)的破壞;同時(shí)在蛋白水平證明17-DMAG能緩解在主動(dòng)脈夾層過(guò)程中平滑肌細(xì)胞的表型轉(zhuǎn)化。結(jié)論:HSP90抑制劑17-DMAG能通過(guò)抑制平滑肌細(xì)胞表型轉(zhuǎn)化減輕主動(dòng)脈組織病變并減少AD模型的死亡率,從而可能成為防治主動(dòng)脈夾層的潛在藥物。
[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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R543.1

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相關(guān)期刊論文 前2條

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本文編號(hào)：1600061