【摘要】 目的比较脑出血(ICH)患者与正常对照者循环miRNA的表达差异,寻找分别与高血压及非高血压ICH相关的标记性miRNA,指导临床诊断与病情评估。找到标记性miRNA的靶标基因/蛋白,明确靶蛋白相关通路,在表观遗传与蛋白水平揭示ICH的发病机制。在人群队列中前瞻性观察标记性miRNA与ICH发生与发展的关系。在已建立的慢病防治社区队列中观察标记性miRNA与初发ICH的关系,在新募集的ICH患者队列中观察标记性miRNA与ICH预后的关系;为ICH的预防和治疗提供新的靶点。方法ICH患者循环miRNA表达谱。(1)比较3例高血压ICH与3例对照、3例非高血压ICH与3例对照血浆miRNA表达谱的差异,经过初步筛选及Realtime PCR方法复筛,选择表达差异最大的6种miRNA(高血压与非高血压ICH各3种);(2)通过逐步扩大样本多次验证的方法,选择2种差异最大的miRNA(高血压与非高血压ICH各1种(以下2种miRNA命名为miR-Xs));(3)分别在社区来源的慢病防治队列及医院来源的ICH队列中前瞻性观察基线血浆miR-Xs水平与新发ICH及ICH预后的关系。蛋白质组差异表达寻找miR-X的靶标基因:(1)选择miR-Xs模拟物(pre-miR-Xs)和miRNA阻遏物(anti-miR-Xs)瞬时转染细胞;(2)蛋白质组分析结合生物信息学靶点预测,选择差异表达量最大的2~4蛋白列入后续研究;(3)将pre-miR-Xs和anti-mir-Xs转染细胞,参照蛋白质结构和功能预测软件分析数据并结合Western blot检测结果,最终选择2种列入后续研究,命名为蛋白Ys。miR-Xs及其靶标基因表达产物的功能与机制研究:(1)通过pre-miR-Xs和anti-miR-Xs瞬时转染后,观察miRNA对细胞功能的影响及作用机制;(2)通过构建蛋白Ys的病毒表达载体,观察miRNA靶标基因表达产物-蛋白Ys对细胞功能的影响及作用机制。结果 (1)通过病例对照研究,发现ICH患者循环miR-26a表达升高,提示miR-26a可能参与了ICH的病理生理过程;(2)确证PEX13是miR-26a的靶基因;(3)miR-26a/PEX13可能通过PDGF-DD通路调节血管平滑肌细胞表型转化、迁移及增殖影响ICH的病理生理过程。结论本文发现miR-26a调控平滑肌细胞表型转化、迁移与增殖。为ICH的病理生理机制研究提供了创新性的研究思路。更多还原

【Abstract】 Objective To compare the miRNA expression differences between intracerebral hemorrhage(ICH) patients and normal controls, and to find marker miRNAs associated with hypertension and non-hypertensive ICH, to guide the clinical diagnosis and disease evaluation. Identify the target gene/protein of the marker miRNA, clarify the pathways related to the target protein, and reveal the pathogenesis of ICH at epigenetic and protein levels. Objective to observe the relationship between marker miRNAs and the occurrence and development of ICH in cohort. To observe the relationship between marker miRNAs and primary ICH in established chronic disease control community cohort → The relationship between the marker miRNA and the prognosis of ICH was observed in the cohort of newly recruited ICH patients → For the prevention and treatment of ICH to provide a new target. Method(1) Hospital Source Stroke Cases-Control Population: From January 2013 to December 2013, From the Second People’s Hospital of Liaocheng inpatients admitted to the ICH and the control group of one hundred cases(including hypertensive and non-hypertensive ICH in fifty cases), To be completed by the end of 2014 2-year followup;(2) Circulation miRNA expression profile in patients with ICH. Comparison of three cases of hypertensive ICH and 3 cases of control, Differences of miRNA expression profiles between three cases of non-hypertensive ICH and three cases of control, after initial screening and realtime PCR method re-screening, six miRNAs with the highest expression difference were selected(hypertension and non-hypertensive ICH of the three kinds. By gradually expanding the sample multiple verification method, two miRNAs with the largest difference were selected(hypertension and nonhypertensive ICH)(hereinafter, these two kinds of miRNAs are named as miR-Xs). The levels of miR-Xs in baseline andplasma levels of ICH and prognosis of ICH were observed and analyzed prospectively in cohort of community-based chronic-disease-control cohort and hospital;(3) Differentially expressed proteins targeted miR-X Target genes. Transient transfection of cells with miR-Xs mimics(pre-miR-Xs) and miRNA repressors(anti-miR-Xs). Proteomics analysis combined with bioinformatics target prediction, The 2-4 proteins with the highest differential expression were selected for further study; Pre-miR-Xs and anti-mir-Xs were transfected into cells, The data were analyzed with reference to protein structure and function prediction software combined with Western blot detection results, two final selections were included in the follow-up study, designated as protein Ys;(4) Study on the function and mechanism of miR-Xs and Its target gene expression products. This study includes two levels of research :By transient transfection of pre-miRXs and anti-miR-Xs, by transient transfection of pre-miR-Xs and anti-miR-Xs. By constructing a viral expression vector for protein Ys, To observe the effect of miRNA target gene expression product Ys on cell function and its mechanism. Results(1) Through case-control study, the expression of miR-26 a increased in patients with ICH, suggesting that miR-26 a may be involved in the pathophysiology of ICH;(2) It was confirmed that PEX13 is the target gene of miR-26 a.(3) Mi R-26 a/PEX13 may regulate the phenotypic transformation, migration and proliferation of vascular smooth muscle cells through PDGF-DD pathway and affect the pathophysiological process of ICH. Conclusion We found that miR-26 a regulates the phenotype transformation, migration and proliferation of smooth muscle cells. It provides an innovative research idea for the pathophysiological mechanism of ICH, and has important scientific significance. At the same time,the results of this research are beneficial to the discovery of related targets and drug development, which is also a prerequisite and important component of cardiovascular and cerebrovascular diseases prevention and control. It has important potential social application value.更多还原