长链非编码RNA lnc-RP11-159G9.5.1-2在颈动脉斑块稳定性中的作用及机制研究

发布时间：2018-05-07 02:22

本文选题：颈动脉狭窄 + 斑块稳定性　；参考：《第二军医大学》2017年硕士论文

【摘要】：研究背景随着人民生活水平的提高和人口结构的老龄化,动脉粥样硬化的发病率和病死率逐年上升,颈动脉狭窄(Carotid artery stenosis)由于易引起脑血管事件而被广泛关注。颈动脉狭窄起病隐匿,常因体检被发现或已经发生临床症状后才被察觉,往往因为没有及时处理而导致脑卒中、偏瘫、失语甚至死亡等严重并发症。近年来颈动脉狭窄的治疗技术取得了明显进步,但仍有很多患者是因为产生了症状并导致并发症才就诊,特别是斑块不稳定的患者,即使狭窄程度不高也很有可能发生脑卒中等严重的并发症。因此,探究颈动脉狭窄的发病和转归机制,寻找与颈动脉斑块稳定性相关的生物标记物,明确其治疗可能的作用靶点,对于该疾病的早期诊断和预防、病情评估和综合治疗都具有重要意义。长链非编码RNA(lncRNA)是指长度大于200nt的不具有编码功能的RNA。过去认为lncRNA是没有任何作用的RNA,随着研究的深入,认为lncRNA在调节组织新陈代谢和病理生理过程中扮演着重要的角色。同时研究也发现,lncRNA与血管疾病密切相关,例如内皮细胞表达的lncRNA(MALAT1和Tie-1-AS)可以调控血管的增生和细胞功能。其中在lnc RNA和动脉粥样硬化性疾病的研究中,关注热点仍为动脉粥样硬化的发生,对lncRNA影响斑块稳定性的机制研究在国内外尚为空白。研究目的明确影响颈动脉斑块稳定性的差异lncRNA;探讨lnc-RP11-159G9.5.1-2对于斑块稳定性的影响;并进一步阐明lnc-RP11-159G9.5.1-2对于调控斑块平滑肌细胞功能的细胞分子机制。研究方法(1)临床样本研究:在通过医院伦理学审查和患者知情同意的前提下,统计本中心2015年8月-2016年6月以颈动脉狭窄为主诉住院治疗并行颈动脉内膜剥脱术的患者,收集颈动脉斑块标本。HE染色将收集的斑块标本区分为稳定性斑块组和不稳定性斑块组,通过高通量芯片筛选两组差异的lncRNA和mRNA表达谱,进而将筛选的差异基因进行生物信息学分析,预测其可能的功能和生物学通路。(2)芯片结果验证:对芯片检测出差异表达的lncRNA进行real-time PCR筛选验证。经过筛选,选择lnc-RP11-159G9.5.1-2作为研究的目标lncRNA,通过共表达分析和GO分析对lnc-RP11-159G9.5.1-2进行生物信息学预测。(3)细胞分子研究:包装干预细胞内lnc-RP11-159G9.5.1-2上调和下调表达的慢病毒,通过慢病毒转染,将血管平滑肌细胞分为lnc-RP11-159G9.5.1-2上调组、lnc-RP11-159G9.5.1-2上调阴性对照组,lnc-RP11-159G9.5.1-2下调组、lnc-RP11-159G9.5.1-2下调阴性对照组和空白对照组,通过cck8比色法法检测lnc-RP11-159G9.5.1-2对平滑肌细胞的增殖功能的影响;通过流式细胞仪Annexin V FITC-PI检测lnc-RP11-159G9.5.1-2对平滑肌细胞的凋亡功能的影响。结果(1)临床样本研究:2015年8月-2016年6月,共收集在长海医院血管外科中心行CEA手术治疗患者标本8例,通过高通量lncRNA基因芯片检测颈动脉稳定性斑块患者和不稳定性斑块患者斑块组织中lncRNA的表达,首次发现了差异表达的212种lncRNA,相较于稳定组斑块,不稳定斑块中115种lncRNA表达上调,97种lncRNA表达下调。通过生物信息学分析,在GO分析和KEGG pathway分析中,差异表达的mRNA在调控血管大小、涉及凋亡的执行阶段的细胞组分解离、血小板聚集、细胞对血管内皮生长因子刺激的反应、负向调节凋亡过程、血管内皮生长因子受体信号通路、正向调节内皮细胞趋化性以及平滑肌细胞迁移等条目中均有富集,这些生物学功能涉及到颈动脉斑块的发生、发展和整个细胞周期过程。(2)芯片结果验证:芯片结果表明lnc-RP11-159G9.5.1-2在不稳定斑块的患者中高表达,通过RT-PCR验证了lnc-RP11-159G9.5.1-2在不稳定斑块患者中相对表达量为4.666±1.741,在稳定性斑块患者中相对表达量为1.000±0.613,不稳定斑块lnc-RP11-159G9.5.1-2含量明显高于稳定性斑块组(p0.0001)。(3)细胞分子研究:lnc-RP11-159G9.5.1-2可抑制血管平滑肌细胞的增殖,促进血管平滑肌细胞的凋亡。结论应用高通量lncRNA基因芯片方法获得了颈动脉狭窄稳定性斑块患者和不稳定性斑块患者差异的lncRNA表达谱,提示不稳定斑块颈动脉狭窄患者中lnc-RP11-159G9.5.1-2高表达。lnc-RP11-159G9.5.1-2通过抑制平滑肌细胞的增殖同时促进平滑肌细胞的凋亡影响颈动脉斑块的稳定性。提示不稳定斑块患者中lnc-RP11-159G9.5.1-2的高表达抑制了斑块纤维帽中平滑肌细胞的增殖同时促进了凋亡,从而使平滑肌细胞减少,造成斑块纤维帽变薄,易于破裂,最终导致斑块不稳定,更易于发生脑血管事件,从而影响了疾病的进程和预后。
[Abstract]:With the improvement of the people's living standard and the aging of the population structure, the incidence and mortality of atherosclerosis are increasing year by year. The carotid stenosis (Carotid artery stenosis) is widely concerned because it is easy to cause cerebrovascular events. The stenosis of the carotid artery is concealed, often after the physical examination is found or the clinical symptoms have already occurred. It is perceived that there are severe complications such as stroke, hemiplegia, aphasia and even death because of the lack of timely treatment. There has been significant progress in the treatment of carotid stenosis in recent years. However, many patients still have symptoms and lead to complications, especially in patients with unstable plaque, even if the degree of stenosis is not high. Therefore, it is important to explore the pathogenesis and prognosis of carotid artery stenosis, to find the biomarkers related to the stability of carotid artery plaque, and to identify the possible therapeutic targets. It is of great significance for the early diagnosis and prevention of the disease, the evaluation of the disease and the comprehensive treatment. Code RNA (lncRNA) means that RNA., which has no coding function in length more than 200nt, used to think that lncRNA is a RNA that has no effect. As the study goes deep, it is considered that lncRNA plays an important role in regulating the metabolic and pathophysiological processes of tissue. Meanwhile, the study also finds that lncRNA is closely related to vascular diseases, such as endothelial cells. The expression of lncRNA (MALAT1 and Tie-1-AS) can regulate the proliferation and cell function of blood vessels. In the study of LNC RNA and atherosclerotic diseases, the focus of attention is atherosclerosis. The mechanism of the effect of lncRNA on plaque stability is still blank at home and abroad. The purpose of this study is to determine the stability of carotid plaque. Sexual difference lncRNA; explore the effect of lnc-RP11-159G9.5.1-2 on plaque stability; and further clarify the molecular mechanism of lnc-RP11-159G9.5.1-2 for the regulation of plaque smooth muscle cell function. Study methods (1) clinical sample study: on the premise of hospital ethics review and patient informed consent, the statistical center August 2015 Patients with carotid stenosis in June, -2016, were treated with carotid endarterectomy in parallel carotid endarterectomy..HE staining of carotid plaque specimens was collected to distinguish the collected plaque specimens into a stable plaque group and an unstable plaque group. A high throughput chip was used to screen two different lncRNA and mRNA expression profiles, and then the differential genes were screened. Carry out bioinformatics analysis, predict its possible function and biological pathways. (2) chip results verify: real-time PCR screening verification of differentially expressed lncRNA by chip. After screening, select lnc-RP11-159G9.5.1-2 as the target lncRNA for study and GO analysis to lnc-RP11-159G9.5.1-2 by common table analysis and GO analysis. Informatics prediction. (3) cell molecular research: packaging interfered with lnc-RP11-159G9.5.1-2 up and down expression of lentivirus in cells. Through lentivirus transfection, vascular smooth muscle cells were divided into lnc-RP11-159G9.5.1-2 up regulation group, lnc-RP11-159G9.5.1-2 up negative control group, lnc-RP11-159G9.5.1-2 down-regulation group and negative lnc-RP11-159G9.5.1-2 downregulation The effect of lnc-RP11-159G9.5.1-2 on the proliferation of smooth muscle cells was detected by CCK8 colorimetric assay in the control group and the blank control group. The effect of lnc-RP11-159G9.5.1-2 on the apoptosis of smooth muscle cells was detected by the flow cytometry Annexin V FITC-PI. Results (1) clinical sample study: in June of August 2015, a total of long sea medicine was collected. 8 patients were treated with CEA surgery in the center of vascular surgery. The expression of lncRNA in plaque tissues of patients with carotid artery stability and unstable plaque was detected by high throughput lncRNA gene chip. The differential expression of 212 kinds of lncRNA was found for the first time. Compared with the stable plaque, 115 kinds of lncRNA expression in unstable plaques were up-regulated and 97 kinds of lncRNA were up. Down regulation of lncRNA expression. Through bioinformatics analysis, in GO analysis and KEGG pathway analysis, differentially expressed mRNA regulates the size of blood vessels, involving cell group dissociation, platelet aggregation, cell response to vascular endothelial growth factor stimulation, negative regulation of apoptosis process, and vascular endothelial growth factor receptor signal The pathway, positive regulation of endothelial cell chemotaxis and smooth muscle cell migration, is enriched. These biological functions involve the occurrence, development of carotid artery plaque and the whole cell cycle process. (2) chip results show that the chip results show that lnc-RP11-159G9.5.1-2 is highly expressed in patients with unstable plaques and is verified by RT-PCR. The relative expression of lnc-RP11-159G9.5.1-2 in unstable plaque patients was 4.666 + 1.741, the relative expression in the patients with stable plaque was 1 + 0.613, and the lnc-RP11-159G9.5.1-2 content of unstable plaque was significantly higher than that of the stable plaque group (P0.0001). (3) cell molecular study: lnc-RP11-159G9.5.1-2 could inhibit the increase of vascular smooth muscle cells. Colonization, promoting apoptosis of vascular smooth muscle cells. Conclusion high throughput lncRNA gene chip method has been used to obtain lncRNA expression profiles in patients with carotid stenosis and stability plaques and unstable plaque patients. It is suggested that lnc-RP11-159G9.5.1-2 high expression.Lnc-RP11-159G9.5.1-2 in patients with unstable plaque carotid artery stenosis can inhibit the smooth muscle by inhibiting smooth muscle cells. The proliferation of cells also promotes the apoptosis of smooth muscle cells and affects the stability of carotid plaque. It is suggested that the high expression of lnc-RP11-159G9.5.1-2 in patients with unstable plaques inhibits the proliferation of smooth muscle cells in the plaques and promotes apoptosis, thus reducing the smooth muscle cells, causing the plaque to be thinner and prone to rupture. It causes plaque instability and is more prone to cerebrovascular events, thereby affecting the progression and prognosis of the disease.

【学位授予单位】：第二军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R543.4

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