基于iTRAQ技术的小鼠缺血性心肌损伤比较蛋白质组学及维生素D结合蛋白在心肌损伤中的作用初探

发布时间：2018-03-21 07:38

本文选题：缺血性心肌损伤　切入点：细胞凋亡　出处：《新疆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:本研究旨在探寻小鼠急性心肌梗死(AMI)后心室重塑早期蛋白质组学的变化,从蛋白质组层面研究心肌梗死后参与心室重塑的分子事件,为心肌梗死后早期心肌保护提供新的思路:1)采用相对和绝对定量的等量异位标签(iTRAQ)技术,高通量、高效率地鉴定及筛选小鼠AMI后差异蛋白的表达;2)在动物模型水平,采用分子生物学手段进行候选靶蛋白的表达验证;3)在细胞模型水平,应用基因转染技术进行靶蛋白的功能验证,探讨靶蛋白的分子生物学作用。方法:1)将雄性实验小鼠随机分为4组,分别为假手术-3天组(Sham-3d)、假手术-7天组(Sham-7d)、心肌梗死-3天组(MI-3d)以及心肌梗死-7天组(MI-7d)组。采用结扎小鼠左冠状动脉的方法建立AMI模型。建模后通过超声心动图、血流动力学以及病理组织学检测方法评估小鼠AMI后心功能及心室重塑情况。提取蛋白质,采用iTRAQ技术进行AMI后3天和7天差异蛋白质的鉴定及筛选,并通过生物信息学分析,对差异表达蛋白的生物学功能进行初步分析;2)在动物组织水平,采用病理组织学检测,评价AMI后早期心肌纤维化导致心室重塑的情况,并进一步采用qRT-PCR及Western Blot方法对小鼠心肌组织进行靶蛋白的表达验证,选择需要进一步功能验证的靶蛋白;3)在细胞水平,建立缺氧/复氧心肌细胞损伤模型,观察维生素D结合蛋白(VDBP)在心肌细胞损伤0h,6h,12h,24h,48h以及72h的表达变化。进一步构建质粒瞬时转染H9C2心肌细胞以过表达VDBP,并采用MTS法、Tunel法及Annexin V/PI法检测细胞凋亡与坏死,采用qRT-PCR及Western Blot方法检测VDBP相关因子的mRNA转录水平及蛋白表达水平变化,探讨VDBP在心肌损伤早期的分子作用。结果:1)成功建立小鼠AMI模型,MI-3d组、MI-7d组心肌梗死面积分别为45.29±7.52%、44.83±7.36%,MI-3d组与MI-7d组左心室重量/胫骨长度均较Sham组增高(P0.001);MI-7d组肺脏重量/胫骨长度较Sham-7d组增高(P0.001)。超声心动图检测显示,与Sham-3d组相比,MI-3d组左心室内径及舒张期外径显著增大(P0.01),左心室前壁及后壁厚度变薄(P0.001),左室短轴缩短率显著下降(P0.001);与Sham-7d组相比,MI-7d组左心室内径及舒张期外径显著增大,左心室前壁及后壁厚度变薄,左室短轴缩短率显著下降(P0.001)。血流动力学测定显示,与Sham-3d组相比,MI-3d组收缩压、平均主动脉压、左心室压及左心室压力变化速率下降(P0.05);与Sham-7d组相比,MI-7d组收缩压、左心室舒张末压及左心室压力变化速率下降下降(P0.01)。HE染色显示,MI-3d组可见大量心肌细胞坏死,可见炎性细胞浸润;MI-7组可见梗死区出现肉芽组织增生,心肌纤维化表现。采用i TRAQ蛋白质组学技术总共鉴定了2540个蛋白,筛选了AMI后表达显著变化的776个差异蛋白。通过对差异蛋白的聚类分析、GO富集及KEGG通路分析,发现wnt、内质网应激(ERS)、维生素D结合蛋白(VDBP)以及肌动蛋白(actin)相关信号通路可能参与AMI后心室重塑的分子生物学过程,其中VDBP可能参与其余3条信号通路的分子生物过程,可能在AMI后心室重塑分子机制中发挥重要作用;2)采用Masson染色发现AMI后心肌胶原含量增加,进一步行免疫组化染色检测I型和III型胶原含量,发现两种类型的胶原在AMI后均增高(P0.01)。通过qRT-PCR检测AMI后心肌损伤因子,与Sham组相比,MI组LDH、cTNT、BNP的mRNA转录水平上调(P0.05)。对靶蛋白信号通路中的关键因子进行qRT-PCR检测,发现在AMI后3天、7天wnt、ERS、VDBP以及actin相关信号通路活化。采用Western blot检测MI组建模后心梗区和Sham组小鼠左心室组织,结果可见VDBP,维生素D受体(VDR)和凝溶胶蛋白(GSN)的蛋白表达水平较假手术组明显上调(P0.001),证实VDBP在AMI后出现蛋白表达的差异性变化;3)成功构建缺氧/复氧H9C2心肌细胞损伤模型,发现VDBP在缺氧/复氧处理后6h、12h的H9C2中表达上调(P0.001)。构建过表达VDBP质粒及对照质粒,并采用脂质体介导的方法瞬时转染H9C2,采用qRT-PCR及Western Blot方法检测VDBP的表达显示,过表达VDBP转染H9C2后,VDBP的mRNA转录水平及蛋白表达水平明显上调(P0.001)。过表达VDBP组经缺氧/复氧6h处理后,细胞凋亡和坏死增加(P0.001)。过表达VDBP处理组的VDR的转录水平及蛋白表达水平明显下调(P0.01),GSN,Caspase3的转录水平及蛋白表达水平上调(P0.001)。结论:1)本研究采用iTRAQ蛋白质组学技术高通量鉴定差异蛋白并筛选,通过分子生物学技术验证,发现wnt、ERS、VDBP以及actin相关信号通路参与AMI后心室重塑的分子生物学过程;2)采用过表达VDBP转染H9C2心肌细胞的方法,在缺氧/复氧心肌细胞损伤模型中进行功能验证,证明VDBP加重心肌细胞损伤的作用,并发现过表达VDBP后抑制VDR、上调GSN活性的分子现象;3)在缺氧/复氧心肌细胞损伤模型中,过表达VDBP后caspase 3的蛋白表达水平上调,证明VDBP可能通过caspase 3介导的细胞凋亡参与AMI后早期心肌细胞损伤的病理生理机制,为靶向干预AMI后心室重塑提供了新的思路。
[Abstract]:Objective: the purpose of this study is to explore the mouse acute myocardial infarction (AMI) on ventricular remodeling after early proteomics changes from the proteome level study of molecular events after myocardial infarction in the early ventricular remodeling, myocardial protection and provide a new way of thinking after myocardial infarction: 1) using isobaric tags for relative and absolute quantitation (iTRAQ) technology, high throughput, high efficiency of the differential expression of protein identification and screening of mice after AMI; 2) in animal model level, to confirm the expression of candidate target proteins by molecular biology; 3) in the cell model, functional verification of target proteins using gene transfection technique, to investigate the molecular biological function of target protein. Methods: 1) male mice were randomly divided into 4 groups, sham operation group were -3 days (Sham-3d), sham operation group (Sham-7d) -7 days -3 days, myocardial infarction group (MI-3d) and myocardial infarction group (MI-7d group) -7 days Methods by ligation of the left coronary artery of mice to establish AMI model. After modeling by echocardiography, hemodynamics and pathological evaluation of detection methods in AMI mice after cardiac function and ventricular remodeling. The extraction of protein was AMI after 3 days and 7 days difference of protein identification and screening by iTRAQ technology, and through the analysis of bioinformatics the biological function of protein expression, preliminary analysis of the difference; 2) in animal tissues, using pathological detection, evaluation of AMI early after myocardial fibrosis leads to ventricular remodeling, and the expression of Western and qRT-PCR by one step Blot method for target protein on myocardial tissue of mice to select target protein for further verification, functional verification 3); at the cellular level, the establishment of reoxygenation myocardial cell injury model of hypoxia / observation of vitamin D binding protein (VDBP) in myocardial injury of 0h cells, 6h, 12h 24h, 48h and 72h, changes of expression. Further constructed plasmids were transiently transfected into H9C2 cells the expression of VDBP in myocardium, and using the method of MTS, apoptosis and necrosis of Tunel method and Annexin V/PI assay, the expression of mRNA mRNA and protein by qRT-PCR and Western Blot VDBP method for the detection of related factors, to explore the VDBP in the early the molecular mechanism of myocardial injury. Results: 1) successfully established a rat model of AMI, MI-3d group, MI-7d group of myocardial infarction area were 45.29 + 7.52%, 44.83 + 7.36%, MI-3d group and MI-7d group, left ventricular weight / tibia length were higher than those in group Sham (P0.001); group MI-7d lung weight / tibia length compared with the Sham-7d group increased (P0.001). Echocardiography showed that compared with the Sham-3d group, MI-3d group, left ventricular diameter and diastolic diameter increased significantly (P0.01), left ventricular anterior wall and posterior wall thickness (P0.001), left ventricular fractional shortening decreased significantly (P0.001); compared with Sham-7d group, MI-7d group, left ventricular diameter and diastolic diameter increased significantly, left ventricular anterior wall and posterior wall thickness, left ventricular fractional shortening decreased significantly (P0.001). Hemodynamic measurements showed that compared with the Sham-3d group, MI-3d group, systolic blood pressure, mean arterial pressure, left changes of ventricular pressure and left ventricular pressure drop rate (P0.05); compared with Sham-7d group, MI-7d group, systolic blood pressure, left ventricular end diastolic pressure and left ventricular pressure changing rate of decline (P0.01).HE staining showed that MI-3d group showed a lot of myocardial cell necrosis, inflammatory cells infiltration; MI-7 group showed granulation appeared in infarction area tissue, myocardial fibrosis. Using I TRAQ proteomics technology in identification of 2540 proteins, 776 differentially expressed proteins were significantly changed after AMI. By clustering the differential protein analysis, pathway enrichment analysis and KEGG GO, found w NT, endoplasmic reticulum stress (ERS), vitamin D binding protein (VDBP) and actin (actin) signaling pathway may be involved in the molecular biology of ventricular remodeling after AMI process, VDBP may be involved in the rest of the 3 signal pathways and molecular biological processes which may play an important role in the molecular mechanism of myocardial remodeling after AMI in 2;) using Masson staining indicated that AMI after myocardial collagen content increased further, immunohistochemical staining for detection of type I and type III collagen content, found two types of collagen were increased after AMI (P0.01). The heart muscle damage factor qRT-PCR detection after AMI, compared with Sham group, MI group, LDH, cTNT, BNP the transcription level of mRNA increased (P0.05). QRT-PCR was detected in the key factor of target protein signaling, found in AMI after 3 days and 7 days of Wnt, ERS, VDBP and actin related signaling pathway by Western blot detection of MI and Sham after establishing the model of myocardial infarction Mice left ventricular tissue, the results showed VDBP, vitamin D receptor (VDR) and gelsolin (GSN) protein expression level was significantly increased compared with the sham operation group (P0.001), confirmed that the VDBP difference of the protein expression changes after AMI; 3) the successful construction of H9C2 reoxygenation injury heart muscle cell model of hypoxia / VDBP, found in hypoxia / reoxygenation after 6h, upregulating the expression of 12h H9C2 (P0.001). Construction of VDBP over expression plasmid and control plasmid, and using the method of liposome mediated transient transfection of H9C2 expression by qRT-PCR and Western Blot method for detection of VDBP showed that over expression of VDBP after transfection of H9C2 expression. The transcription level of mRNA VDBP and protein levels were significantly up-regulated (P0.001). The expression of VDBP after hypoxia / reoxygenation after 6h treatment, cell apoptosis and necrosis increased (P0.001). The expression levels of mRNA and protein expression in VDBP group VDR was significantly reduced (P0.01), GSN, Caspase3 The expression of mRNA and protein level increased (P0.001). Conclusion: 1) this research uses iTRAQ technologies for high-throughput proteomics screening and identification of differential proteins, through molecular biology technology verification, found that Wnt, ERS, VDBP and molecular biology actin related signal pathway in AMI after ventricular reshaping process; 2) by over expression methods H9C2 myocardial cells transfected with VDBP, functional verification on reoxygenation myocardial cell injury model in hypoxia / VDBP, increase the myocardial cell injury, and have been found to inhibit the expression of VDR VDBP, part of the increase of GSN activity of the phenomenon; 3) in hypoxia reoxygenation myocardial cell injury model / overexpression of VDBP. After the expression of caspase 3 protein level increased, prove the pathophysiological mechanism of early myocardial cell damage in VDBP cells apoptosis through caspase mediated by AMI in 3, targeting the ventricular remodeling after the intervention of AMI provides a new Thinking.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R542.22

【相似文献】