A型急性主动脉夹层的分子机制研究与预后评价

发布时间：2018-06-23 22:46

  本文选题：急性主动脉夹层 + 基因芯片　； 参考：《北京协和医学院》2015年博士论文

【摘要】：第一部分A型急性主动脉夹层主动脉组织差异表达基因的筛选和验证摘要目的：急性主动脉夹层(Acute aortic dissection, AAD)是临床常见的危急重症,具有较高的致死性,但其分子机制尚未阐明。本研究通过全基因组转录谱芯片分析筛选AAD患者病变主动脉组织和正常对照主动脉组织差异表达的基因,通过生物信息学分析挑选核心差异表达基因在蛋白水平进行验证,为后续寻找新的有关主动脉夹层发生的重要信号传导途径和重要基因及探讨这些基因在AAD发生、发展中的作用和机制奠定基础。方法：采用标准的组织收集方法分别收集8例AAD患者和8例正常器官捐献者的升主动脉标本。AAD患者和正常对照各选取4例升主动脉组织标本进行全基因组表达谱芯片分析,筛选AAD患者和正常对照主动脉组织差异表达的基因,利用生物信息学分析差异表达的基因可能的生物学功能,根据生物信息学分析结果挑选可能在AAD发生、发展中具有重要作用的信号传导通路和核心基因；然后提取8例AAD患者和8例正常对照的主动脉组织蛋白,通过Western blotting验证mRNA表达明显差异的核心基因在AAD患者和正常对照主动脉组织中的蛋白表达差异；由于转化生长因子-β(Transform growth factor-β, TGF-β)信号通路是已知的与AAD发生相关的分子通路,同时在8例AAD患者和8例正常对照主动脉组织中通过Western blotting分析了TGF-β通路中关键分子SMAD2和SMAD3的蛋白表达是否有差异。结果：(1)基因表达谱芯片分析显示,AAD患者病变主动脉组织与正常对照主动脉组织比较,mRNA表达差异大于2倍的基因有129个,其中83个基因表达上调,46个基因表达下调。生物信息学分析提示,差异表达的基因主要涉及粘附斑(FN1、ITGA9、PAK7、C0L6A3、FLNC)和肌动蛋白骨架调节(FN1、ITGA9、PAK7、PIP5K1B, RRAS2)两条信号通路,其中ITGA9基因是编码整合素α9亚基的基因,在两条信号传导通路中具有关键作用,表达下调达50%。因此本研究挑选了ITGA9作为可能与AAD发病相关的核心关键基因；(2)Western blotting分析证实,AAD患者主动脉组织中ITGA9的表达显著低于正常对照(P=0.003)；(3)Western blotting分析显示,AAD患者病变主动脉组织中磷酸化SMAD2 (p-SMAD2)表达下调(P0.05),而总SMAD2、总SMAD3、磷酸化SMAD3(p-SMAD3)和正常对照无显著差异(P值均0.05)。结论：通过组织表达谱芯片分析发现AAD患者病变主动脉组织和正常对照主动脉组织的基因表达谱有较大差异。生物信息分析显示,主动脉夹层病变组织中表达明显差异的两条通路是粘附斑和肌动蛋白骨架调节相关通路,其中ITGA9基因是同时参与两条通路的核心基因；在蛋白水平证实AAD患者病变主动脉组织的ITGA9表达降低,而传统TGF-β信号通路中的p-SMAD2蛋白表达水平在AAD患者中显著降低,但其他关键成分无明显变化,提示ITGA9基因可能是参与AAD发生的重要基因,需要进一步的功能研究验证其在AAD发生发展中的作用；p-SMAD2是否与ITGA9相互作用参与AAD发病机制尚需要进一步实验研究。第二部分ITGA9对大鼠主动脉血管平滑肌细胞表型的影响摘要目的：主动脉中层退行性变是目前被广为认同的主动脉夹层的主要发病机制,但其确切的分子机制尚不明确。血管平滑肌细胞(Vascular smooth muscle cell, VSMC)是维持主动脉正常结构和功能的主要成分。VSMC有收缩型和合成型两种表型,VSMC的表型转换是主动脉退行性变的分子机制之一。前期实验中我们通过基因芯片和蛋白水平验证发现整合素α9亚基的编码基因ITGA9在AAD患者病变主动脉组织中表达显著低于正常对照。由于ITGA9在主动脉VSMC胞膜表达,且同时参与细胞粘附和骨架蛋白调节通路,因此推测ITGA9在AAD的发病中可能具有重要作用。本实验部分拟通过细胞学实验研究ITGA9表达改变对VSMC表型的影响,探讨ITGA9表达下调在AAD发病中的可能机制。方法：首先通过组织块贴壁法原代培养大鼠主动脉VSMC并证实大鼠主动脉VSMC表达ITGA9;然后将荧光标记的小分子RNA探针转染VSMC,观察小分子RNA在大鼠VSMC中的转染效率；明确小分子RNA可转染大鼠VSMC后,针对大鼠ITGA9基因设计特异性的短链干扰RNA (Short interfering RNA, siRNA),转染大鼠VSMC敲低VSMC ITGA9的表达,Western blotting检测ITGA9敲低后VSMC收缩型标志物(SM22α和α-SMA)和合成型标志物(OPN和SMemb)表达变化以及细胞划痕试验观察ITGA9敲低后VSMC增殖、迁移能力的变化；最后构建腺病毒——ITGA9重组载体,包装腺病毒,感染经血小板源性生长因子(PDGF)诱导呈合成型的VSMC过表达ITGA9,Western blotting检测过表达ITGA9后VSMC收缩型标志物(SM22a和α-SMA)和合成型标志物(SMemb)表达变化。结果：(1)组织块贴壁法成功培养出原代大鼠主动脉VSMC并经免疫荧光所鉴定；(2) Western blotting证实,大鼠主动脉VSMC表达ITGA9,为下一步干预ITGA9的表达奠定了基础；(3)荧光标记的小分子RNA能顺利转染大鼠主动脉VSMC;(4)针对ITGA9设计的三条siRNA,其中一条能高效敲低VSMC中ITGA9的表达；(5)ITGA9敲低后,经Western blotting证实,VSMC收缩型标志物(SM22 α和α-SMA)表达降低,合成型标志物(OPN和SMemb)表达升高；细胞划痕试验显示,ITGA9敲低后VSMC增殖、迁移能力增强,提示VSMC表型从收缩型向合成型转换；(6)成功构建腺病毒-ITGA9重组载体,腺病毒包装后病毒滴度达病毒滴度达1×109PFU/mL;(7)重组腺病毒感染大鼠主动脉VSMC过表达ITGA9后,VSMC收缩型标志物SM22 α显著升高(P=0.017),α-SMA表达也升高(P=0.119), SMemb表达降低(P=0.104),但未到达统计学意义。结论：ITGA9表达改变会引起VSMC表型的转化,ITGA9的低表达可能通过诱导VSMC从收缩型向合成型转换参与AAD的发生。本实验结果提示ITGA9基因表达异常可能参与主动脉夹层发生的分子机制。敲低后,经Western blotting证实,VSMC收缩型标志物(SM22 α和α-SMA)表达降低,合成型标志物(OPN和SMemb)表达升高；细胞划痕试验显示,ITGA9敲低后VSMC增殖、迁移能力增强,提示VSMC表型从收缩型向合成型转换；(6)成功构建腺病毒-ITGA9重组载体,腺病毒包装后病毒滴度达病毒滴度达1×109PFU/mL;(7)重组腺病毒感染大鼠主动脉VSMC过表达ITGA9后,VSMC收缩型标志物SM22 α显著升高(P=0.017),α-SMA表达也升高(P=0.119), SMemb表达降低(P=0.104),但未到达统计学意义。结论：ITGA9表达改变会引起VSMC表型的转化,ITGA9的低表达可能通过诱导VSMC从收缩型向合成型转换参与AAD的发生。本实验结果提示ITGA9基因表达异常可能参与主动脉夹层发生的分子机制。第三部分入院时D-二聚体水平对A型急性主动脉夹层患者短期和长期死亡的影响摘要目的：D-二聚体是交联的纤维蛋白降解产物,其升高提示凝血和纤溶系统的激活。先前研究提示D-二聚体可作为辅助诊断或排除急性主动脉夹层(Acute aortic dissection, AAD)的分子标志物,但其作为预后评估因素的研究较少。本研究通过相对大样本的研究评估入院时D-二聚体水平对A型AAD患者住院期间和长期随访死亡的影响,旨在探讨入院时D-二聚体水平对AAD短期和长期预后的预测价值。方法：本研究设计为单中心、前瞻性、观察性研究,研究对象为连续入选从2010年3月至2011年6月就诊于阜外心血管病医院急诊中心并经CT证实的A型AAD患者。排除标准为主动脉壁内血肿、主动脉透壁溃疡、外伤性主动脉破裂、医源性夹层、妊娠、马凡综合征、家族性胸主动脉夹层等。采集患者入院时的基线资料,检测患者入院时D-二聚体水平,并记录患者住院期间的其他临床资料。主要终点事件为住院期间全因死亡率和长期随访全因死亡率。首先比较存活患者和死亡患者入院时D-二聚体水平的差异；然后按入院时D-二聚体水平四分位分组比较各组患者住院死亡率和长期死亡率；通过单因素和多因素Cox回归分析入院时D-二聚体水平和住院死亡和长期死亡的相关性。由手术治疗对患者预后有显著影响,将患者分为手术治疗组和保守治疗组,比较两组中不同D-二聚体水平(6.10μg/mL和≤6.10 μ g/mL)的患者住院死亡率和长期死亡率的差异。结果：入选2010年3月至2011年6月间就诊于阜外心血管病医院并经主动脉CT检查确诊的AAD患者共375例,其中A型AAD 225例,B型AAD 150例。排除13例患者临床资料不全、失访或符合排除标准,共212例A型AAD进入统计分析。患者中位住院天数14天(25%~75%,9~20天),出院后中位随访时间18.8月(25%-75%,6.7-24.4月)。患者平均年龄48.5±11.5岁,男性比例较高(75.9%)。住院期间全因死亡率和长期随访全因死亡率分别为12.7%(27/212)和12.4%(23/185)。与存活者相比,死亡患者入院时D-二聚体水平显著增高(中位数6.8 vs.2.5 μg/mL, P0.001)、白细胞计数、肌酐水平也显著高于存活组患者(P值均0.001),但接受手术治疗的比例显著低于存活组患者(14.0% vs.74.7%,P0.001)。将患者按入院时D-二聚体四分位分组(Ql:≤1.06 μg/mL; Q2:1.07~2.82 μg/mL; Q3:2.83-6.10 μg/mL; Q4:6.10μg/mL),第四分位(Q4)组患者住院期间死亡率(30.2%)和长期随访死亡率(24.3%)均显著高于其他组患者(P0.001和P=0.046)。多因素Cox回归分析显示,在校正了其他影响住院期间死亡和长期随访死亡的因素后,入院时D-二聚体6.10 μg/mL是影响A型AAD患者住院期间死亡的独立危险因素(HR=6.124,95%CI 1.345-27.892, P=0.019),但入院时D-二聚体水平和AAD患者长期死亡无显著相关性。是否接受手术治疗是影响AAD患者住院期间死亡和长期随访死亡最主要因素。将患者按是否接受手术治疗分组后,接受手术治疗的患者中,入院时D-二聚体6.10μg/mL和≤6.10μg/mL的患者比较住院期间死亡率和长期随访死亡率无显著差异,而保守治疗的患者中,入院时D-二聚体6.10μg/mL的患者住院期间死亡率和长期随访死亡率均显著高于入院时D-二聚体≤6.10μg/mL的患者。结论:(1)手术治疗是降低A型AAD患者住院期间死亡和长期死亡的主要措施；(2)入院时D-二聚体水平增高(6.10μg/mL)增加住院期间死亡风险,但和长期死亡无相关性；(3)入院时D-二聚体水平对保守治疗的A型AAD患者预后可能有更好的预测价值。
[Abstract]:Selection and validation of differentially expressed genes in aortic dissection of type A acute aortic dissection (Acute aortic dissection, AAD) is a common clinical critical disease, with high lethality, but its molecular mechanism has not been elucidated. This study screened AAD by whole genome transcriptional chip analysis. The gene differentially expressed in the aortic tissue and the normal control of the aorta, selected the core differentially expressed genes through bioinformatics analysis to verify the protein level, in order to find out the important signal transduction pathways and important bases related to the occurrence of aortic dissection and to explore the development of these genes in AAD. Methods: a standard tissue collection method was used to collect 8 cases of AAD patients and 8 normal organ donors in the ascending aorta of.AAD patients and normal controls, and 4 ascending aorta tissue specimens were selected for total genomic chip analysis. The difference between the selected AAD patients and the normal control aorta was poor. Differentially expressed genes, using bioinformatics to analyze the possible biological functions of differentially expressed genes, select the signal transduction pathways and core genes that may play an important role in the development of AAD according to the bioinformatics analysis results, and then extract 8 cases of AAD and 8 normal controls of the aortic tissue protein, through Western Blotting verifies the difference in the protein expression in the AAD patients and the normal control aorta, while the mRNA signaling pathway is known to be a known molecular pathway associated with AAD, and in 8 cases of AAD and 8 normal controls. Western blotting was used to analyze the difference in the protein expression of SMAD2 and SMAD3 key molecules in TGF- beta pathway. Results: (1) gene expression chip analysis showed that there were 129 genes in the AAD patients' aorta and the normal control aorta, and the expression of mRNA was more than 2 times, of which 83 genes were up-regulated and 46. The bioinformatics analysis suggests that the differentially expressed genes are mainly involved in two signaling pathways: FN1, ITGA9, PAK7, C0L6A3, FLNC, and actin cytoskeleton regulation (FN1, ITGA9, PAK7, PIP5K1B, RRAS2), of which ITGA9 gene is the gene encoding integrin 9 subunit, which plays a key role in the two signal transduction pathways. In this study, ITGA9 was selected as a key key gene for the pathogenesis of AAD, and (2) Western blotting analysis confirmed that the expression of ITGA9 in the aorta of AAD patients was significantly lower than that of the normal control (P=0.003); (3) Western blotting analysis showed that the phosphorylation SMAD2 in the pathological aorta tissues of AAD patients. There was no significant difference in expression (P0.05), while total SMAD2, total SMAD3, phosphorylated SMAD3 (p-SMAD3) and normal controls were not significantly different (P value was 0.05). Conclusion: the gene expression profiles of the aorta and normal control aorta of the patients with AAD were significantly different by tissue expression chip analysis. Bioinformatics analysis showed that the aortic dissection was dissecting. The two pathways that express distinct differences in the tissues are the regulatory pathway of the adhesion and actin framework, in which the ITGA9 gene is the core gene involved in the simultaneous participation of two pathways, and the protein level indicates that the ITGA9 expression in the pathological aorta of the patients with AAD is reduced, while the level of p-SMAD2 protein expression in the traditional TGF- beta signaling pathway is in AAD patients. The ITGA9 gene may be an important gene involved in the occurrence of AAD. It needs further functional study to verify its role in the development of AAD, and the interaction of p-SMAD2 with ITGA9 to participate in the pathogenesis of AAD still needs further experimental study. The second part of ITGA9 is active to rats. Objective: the effect of the phenotype of vascular smooth muscle cells (Vascular smooth muscle cell, VSMC) is the major component of the normal structure and function of the aorta,.VSM, the main pathogenesis of aortic dissection, which is widely recognized. C has two types of phenotype of contractile and synthetic type. The phenotypic conversion of VSMC is one of the molecular mechanisms of the degeneration of the aorta. In previous experiments, we found that the encoding gene ITGA9 of the integrin alpha 9 subunit was significantly lower than the normal control in the diseased aorta of AAD patients. As ITGA9 is in the aorta VS MC membrane expression, and also involved in cell adhesion and cytoskeleton regulation pathway, thus speculates that ITGA9 may play an important role in the pathogenesis of AAD. This experiment is to study the effect of ITGA9 expression on the VSMC phenotype by cytological experiments and to explore the possible mechanism of ITGA9 expression down regulation in AAD hair disease. The rat aorta VSMC was cultured and VSMC expressed ITGA9 in rat aorta, and then ITGA9 was expressed in rat aorta. Then VSMC was transfected by small molecule RNA probe labeled with fluorescence. The transfection efficiency of small molecule RNA in rat VSMC was observed. After the small molecule RNA could be transfected to rat VSMC, the specific short chain interference RNA (Short) aimed at the rat ITGA9 gene was designed. Ng RNA, siRNA), transfection of VSMC knockout low VSMC ITGA9 expression in rats, Western blotting detected VSMC contractile markers (SM22 alpha and alpha -SMA) and synthetic markers, and cell scratch test. VSMC overexpressed ITGA9 was induced by platelet derived growth factor (PDGF), and Western blotting was used to detect VSMC contractile markers (SM22a and -SMA) and synthetic markers (SMemb) after ITGA9 expression. Results: (1) tissue block adherence method successfully cultured primary rat aorta and was immune Identified by pestilence fluorescence; (2) Western blotting confirmed that VSMC expressed ITGA9 in rat aorta, which laid the foundation for further intervention in ITGA9 expression; (3) the fluorescent labeled small molecule RNA could successfully transfect VSMC in rat aorta; (4) three siRNA designed against ITGA9, one of which could knock low VSMC ITGA9 expression; (5) ITGA9 was low, The expression of VSMC contractile markers (SM22 and alpha -SMA) was reduced by Western blotting, and the expression of synthetic markers (OPN and SMemb) increased. The cell scratch test showed that the VSMC proliferation and migration ability increased after the knockout of ITGA9, suggesting that the VSMC phenotype changed from contractile to synthetic; (6) the adenovirus -ITGA9 recombinant vector was successfully constructed, adenovirus package was successfully constructed. After loading, the titer of virus titer reached 1 x 109PFU/mL; (7) after VSMC overexpressed ITGA9 in rat aorta of recombinant adenovirus, SM22 alpha of VSMC contraction marker increased significantly (P=0.017), the expression of alpha -SMA increased (P=0.119), SMemb expression decreased (P=0.104), but it was not statistically significant. Conclusion: ITGA9 expression change will cause the VSMC phenotypes to turn. The low expression of ITGA9 may be involved in the occurrence of AAD by inducing the convergent transformation of VSMC from contractile type. The results of this experiment suggest that abnormal ITGA9 gene expression may be involved in the molecular mechanism of aortic dissection. After the knock down, the expression of VSMC contraction markers (SM22 A and alpha -SMA) is reduced by Western blotting, and the synthetic marker (OPN and VSMC) The expression of SMemb increased, and the cell scratch test showed that the VSMC proliferation and migration ability increased after the ITGA9 knockout, suggesting that the VSMC phenotype was converted from contractile to co forming; (6) the recombinant adenovirus -ITGA9 recombinant vector was successfully constructed. The virus titer of the adenovirus was 1 * 109PFU/ mL after the adenovirus package, and (7) the recombinant adenovirus infected rat aorta VSMC overexpressed ITGA. After 9, the SM22 alpha of VSMC contraction marker increased significantly (P=0.017), the expression of alpha -SMA increased (P=0.119) and SMemb expression decreased (P=0.104), but it did not reach statistical significance. Conclusion: the change of ITGA9 expression may cause the transformation of the VSMC phenotype, and the low expression of ITGA9 may be involved in the occurrence of AAD, by inducing VSMC contractile type transformation. Results suggest that abnormal ITGA9 gene expression may participate in the molecular mechanism of aortic dissection. Third the effect of D- two polymer level on short-term and long-term death of patients with type A acute aortic dissection at admission: D- two polymer is a cross-linked fibrin degradation product, and its elevation suggests activation of coagulation and fibrinolytic system. The study suggests that D- two polymer can be used as a molecular marker for the diagnosis or exclusion of acute aortic dissection (Acute aortic dissection, AAD), but it is less studied as a prognostic factor. This study evaluated the effects of D- two polymer levels at admission to patients with A AAD during hospitalization and long-term follow-up by relatively large sample studies. The purpose of this study was to explore the predictive value of D- two polymer level on the short-term and long-term prognosis of AAD. Methods: This study was designed as a single center, prospective, observational study. The object of this study was to be selected from March 2010 to June 2011 in the emergency center of Fuwai Hospital of Cardiovascular Disease and the A type AAD patients confirmed by CT. The exclusion criteria were the aorta. Intramural hematoma, transmural ulcer of aorta, traumatic rupture of aorta, iatrogenic interlayer, pregnancy, Marfan syndrome, familial thoracic aortic dissection, etc.. Baseline data of hospitalized patients were collected, D- two polymer levels were detected at admission, and the patient's clinical data were recorded during hospitalization. The main terminal event was the death of all patients during hospitalization. Death rate and long-term follow-up all cause mortality. First, compare the difference in the D- two polymer level between the survivors and the death patients at admission, and then compare the hospitalized mortality and long-term mortality of the patients at the admission of the D- two polymer level Four Division at admission, and the D- two polymer level and hospital death at admission by single factor and multiple factor Cox regression analysis. The correlation between death and long-term death. The surgical treatment had a significant impact on the prognosis of the patients. The patients were divided into surgical and conservative treatment groups. The differences in hospitalization and long-term mortality were compared between the two groups of the two groups (6.10 mu g/mL and < 6.10 g/mL). There were 375 patients with AAD diagnosed by CT in Fuwai Hospital of Cardiovascular Disease, including 225 cases of A AAD and 150 cases of B AAD, excluding 13 patients with incomplete clinical data, missing or conforming to the exclusion criteria, 212 cases of A type AAD entered the statistical analysis. The median hospital days were 14 days (25%~ 75%, 9~20 days), and the median follow-up time after discharge was 18.8 months (25%-75%). The average age of the patients was 48.5 + 11.5 years, and the male ratio was higher (75.9%). The total cause and long-term follow-up mortality of the patients were 12.7% (27/212) and 12.4% (23/185). Compared with the survivors, the level of D- two polymer was significantly higher at admission (median 6.8 vs.2.5, g/mL, P0.001), white blood cell count, creatinine water. The level was significantly higher than that in the survival group (P value was 0.001), but the proportion of surgical treatment was significantly lower than that in the survival group (14% vs.74.7%, P0.001). The patients were divided into four sub groups (Ql: < 1.06 mu g/mL; Q2:1.07~2.82 u g/mL; Q3:2.83-6.10 mu g/mL; Q4:6.10 Mu g/mL), and the mortality rate of the fourth division patients during hospitalization. (30.2%) and long-term follow-up mortality (24.3%) were significantly higher than those in other groups (P0.001 and P=0.046). Multiple factor Cox regression analysis showed that D- two polymer 6.10 mu g/mL was an independent risk factor affecting the death of A type AAD patients during hospitalization after admission to other factors affecting death and long-term follow-up death. CI 1.345-27.892, P=0.019), but there was no significant correlation between the level of D- two polymer at admission and the long-term death of AAD patients. Whether surgical treatment was the most important factor affecting death and long-term follow-up of AAD patients was the most important factor. The patients who were treated with surgical treatment were admitted to the hospital after the operation, and the D- two polymer was 6.10 mu g at admission. Patients with /mL and less than 6.10 g/mL had no significant difference in hospital mortality and long-term follow-up mortality, while patients with conservative treatment had no significant difference.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R543.1
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本文编号：2058747