高迁移率蛋白1通过炎症反应促进心肌肥厚及其机制研究

发布时间：2018-08-15 12:06

【摘要】：背景心肌肥厚和许多心脏疾病有关,包括心肌缺血,瓣膜狭窄,肥厚性心肌病等,是心肌细胞受到内源性刺激或外源性刺激后导致的心脏形态、心室肌及室间隔的厚度等一系列组织结构的重构,最终走向心衰、心律失常、心源性猝死。然而,心肌肥厚的发病机理至今仍未完全阐明,临床亦无有效防治措施。细胞内外信号通路是调节细胞数量和大小的中心环节之一。高迁移率族蛋白1(high mobility group box1,HMGB1)作为新近发现的核蛋白,广泛表达于几乎所有的真核细胞中,主要与晚期糖基化终末受体(receptor for advanced glycation endproducts,RAGE)结合,通过Ras、MAPK和NF-kB信号通路调节基因转录、激活炎症反应。文献报道HMGB1在心肌缺血、心梗、心衰中表达明显增加,与心肌损伤及心室重构有密切关系。HMGB1信号通路是否通过这种方式影响心肌肥厚的发生发展仍不清楚。目的1.将课题前期筛选出的差异性表达基因利用IPA数据分析技术筛选出肥厚型心肌病病人心肌组织最显著激活的信号蛋白。2.在病理水平验证HMGB1在肥厚型梗阻性心肌病病人表达。3.探讨HMGB1在心肌肥厚模型中的表达情况。方法1.收集安贞医院6位严重肥厚型心肌病患者和武汉大学附属人民医院提供的6位供体的心肌组织,从中提取mRNA,进行反转录,利用Illumina测序平台进行转录组测序,得到的数据通过生物信息学方法筛选差异表达基因(课题前期已完成),将筛选得到的差异表达基因输入IPA数据分析平台,检测差异基因在经典信号通路中的富集情况,制作信号通路柱状图,并进行经典通路分析。2.将肥厚型梗阻性心肌病病人心肌和对照组心肌制作病理切片,免疫组化染色,光镜下统计HMGB1表达量。3.制作小鼠肥厚心肌模型(主动脉弓缩窄术),在RNA水平研究HMGB1的表达情况。结果1.利用Illumina测序平台,得到了全部转录组数据,整理差异性基因表达,并且数据良好,可以进行生物信息学分析。通过IPA在线整合分析软件,差异基因所调控的经典通路由IPA所收集归纳的800条信号、代谢通路所总结;所有的信号通路使用-Log(P-value)进行排序,比较Z-score值,Z-score2代表该Pathway被显著激活,Z-score-2代表该Pathway被显著抑制;Ratio表示在此信号通路中差异基因数与信号通路的所有基因数的比值实验数据预测的信号通路展示了实验数据对信号通路的信号传递的影响。在信号通路图中给出了各种分子的信号传递过程和差异基因的上下调情况,通过分子激活预测,对其他基因进行了激活抑制预测。对|Z-score|2的信号通路进行富集程度排名,HMGB1Signaling是在|Z-score|2的信号通路中,显著富集排名第一的信号通路。2.利用免疫组化染色,和正常对照供体心肌相比,肥厚型梗阻性心肌病病人心肌HMGB1表达量增加。3.肥厚模型制备成功,和对照组相比,在mRNA水平手术组小鼠心肌组织HMGB1表达量增加。结论1.本研究在肥厚型梗阻性心肌病中筛选差异性表达基因上行IPA分析和通路预测,得出最显著激活的信号蛋白HMGB1。2.在肥厚型梗阻性心肌病病人心肌中在病理水平验证HMGB1表达量增加。随后通过构建小鼠肥厚模型,在1-2周室间隔最厚,收缩功能最强,肥厚marker表达明显,HMGB1表达量最高。提示早期炎症因子HMGB1可能和心肌肥厚密切相关,并参与超负荷所致的心肌损伤。
[Abstract]:Background Myocardial hypertrophy is associated with many heart diseases, including myocardial ischemia, valvular stenosis, hypertrophic cardiomyopathy and so on. It is the remodeling of cardiac morphology, thickness of ventricular myocardium and septum resulting from endogenous or exogenous stimulation of cardiomyocytes, and eventually leads to heart failure, arrhythmia and sudden cardiac death. High mobility group box 1 (HMGB1), as a newly discovered nuclear protein, is widely expressed in almost all eukaryotic cells. Receptors for advanced glycation end products (RAGE) bind to regulate gene transcription and activate inflammation through Ras, MAPK and NF-kB signaling pathways. It is reported that HMGB1 expression is significantly increased in myocardial ischemia, myocardial infarction and heart failure, which is closely related to myocardial injury and ventricular remodeling. Objective 1. To screen the most prominent signal proteins in myocardial tissue of patients with hypertrophic cardiomyopathy by IPA data analysis. 2. To verify the expression of HMGB1 in patients with hypertrophic obstructive cardiomyopathy at pathological level. 3. To explore the expression of HMG in patients with hypertrophic obstructive cardiomyopathy. Expression of B1 in myocardial hypertrophy model. Methods 1. Myocardial tissues from 6 patients with severe hypertrophic cardiomyopathy in Anzhen Hospital and 6 donors provided by People's Hospital Affiliated to Wuhan University were collected. The mRNA was extracted and retrieved. The transcriptome was sequenced by Illumina sequencing platform. The obtained data were screened by bioinformatics method. Differentially expressed genes (previously completed), the differentially expressed genes were input into the IPA data analysis platform to detect the enrichment of differentially expressed genes in the classical signaling pathway, and then the signal pathway histogram was made and the classical pathway analysis was carried out. 2. The myocardium of patients with hypertrophic obstructive cardiomyopathy and the control group were pathologically cut. The expression of HMGB1 was studied at RNA level. Results 1. Using Illumina sequencing platform, all the transcriptome data were obtained, and the differentially expressed genes were sorted out. The data were good enough for bioinformatics analysis. Through IPA on-line integration analysis software, the classical pathways regulated by differentially expressed genes were summarized by 800 signals collected by IPA, and metabolic pathways were summarized; all signaling pathways were sequenced using - Log (P-value) to compare Z-score values. Z-score 2 represented that the Pathway was significantly activated and Z-score-2 represented that the Pathway was significantly inhibited; Ratio said in this letter. The signal pathway predicted by the ratio of the number of differentially expressed genes to the number of all genes in the signaling pathway shows the effect of the experimental data on the signal transduction of the signaling pathway. HMGB1 signaling is the first signaling pathway significantly enriched in | Z-score | 2 signaling pathway. 2. Immunohistochemical staining showed an increased expression of HMGB1 in hypertrophic obstructive cardiomyopathy compared with normal donor myocardium. Compared with the control group, the expression of HMGB1 in the myocardium of the mice in the mRNA level group increased. Conclusion 1. This study screened differentially expressed genes in hypertrophic obstructive cardiomyopathy and predicted the pathway by IPA analysis. The most significant activation signal protein HMGB1.2 was found in the myocardium of patients with hypertrophic obstructive cardiomyopathy. The expression of HMGB1 was increased at pathological level. Then the mouse model of hypertrophy was constructed, which showed the thickest ventricular septum, strongest contractile function, markeder expression of hypertrophy and the highest expression of HMGB1 at 1-2 weeks.
【学位授予单位】：首都医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R542.2

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