本文選題：頸動(dòng)脈狹窄 + 斑塊穩(wěn)定性　； 參考：《第二軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景隨著人民生活水平的提高和人口結(jié)構(gòu)的老齡化,動(dòng)脈粥樣硬化的發(fā)病率和病死率逐年上升,頸動(dòng)脈狹窄(Carotid artery stenosis)由于易引起腦血管事件而被廣泛關(guān)注。頸動(dòng)脈狹窄起病隱匿,常因體檢被發(fā)現(xiàn)或已經(jīng)發(fā)生臨床癥狀后才被察覺(jué),往往因?yàn)闆](méi)有及時(shí)處理而導(dǎo)致腦卒中、偏癱、失語(yǔ)甚至死亡等嚴(yán)重并發(fā)癥。近年來(lái)頸動(dòng)脈狹窄的治療技術(shù)取得了明顯進(jìn)步,但仍有很多患者是因?yàn)楫a(chǎn)生了癥狀并導(dǎo)致并發(fā)癥才就診,特別是斑塊不穩(wěn)定的患者,即使狹窄程度不高也很有可能發(fā)生腦卒中等嚴(yán)重的并發(fā)癥。因此,探究頸動(dòng)脈狹窄的發(fā)病和轉(zhuǎn)歸機(jī)制,尋找與頸動(dòng)脈斑塊穩(wěn)定性相關(guān)的生物標(biāo)記物,明確其治療可能的作用靶點(diǎn),對(duì)于該疾病的早期診斷和預(yù)防、病情評(píng)估和綜合治療都具有重要意義。長(zhǎng)鏈非編碼RNA(lncRNA)是指長(zhǎng)度大于200nt的不具有編碼功能的RNA。過(guò)去認(rèn)為lncRNA是沒(méi)有任何作用的RNA,隨著研究的深入,認(rèn)為lncRNA在調(diào)節(jié)組織新陳代謝和病理生理過(guò)程中扮演著重要的角色。同時(shí)研究也發(fā)現(xiàn),lncRNA與血管疾病密切相關(guān),例如內(nèi)皮細(xì)胞表達(dá)的lncRNA(MALAT1和Tie-1-AS)可以調(diào)控血管的增生和細(xì)胞功能。其中在lnc RNA和動(dòng)脈粥樣硬化性疾病的研究中,關(guān)注熱點(diǎn)仍為動(dòng)脈粥樣硬化的發(fā)生,對(duì)lncRNA影響斑塊穩(wěn)定性的機(jī)制研究在國(guó)內(nèi)外尚為空白。研究目的明確影響頸動(dòng)脈斑塊穩(wěn)定性的差異lncRNA;探討lnc-RP11-159G9.5.1-2對(duì)于斑塊穩(wěn)定性的影響;并進(jìn)一步闡明lnc-RP11-159G9.5.1-2對(duì)于調(diào)控斑塊平滑肌細(xì)胞功能的細(xì)胞分子機(jī)制。研究方法(1)臨床樣本研究:在通過(guò)醫(yī)院倫理學(xué)審查和患者知情同意的前提下,統(tǒng)計(jì)本中心2015年8月-2016年6月以頸動(dòng)脈狹窄為主訴住院治療并行頸動(dòng)脈內(nèi)膜剝脫術(shù)的患者,收集頸動(dòng)脈斑塊標(biāo)本。HE染色將收集的斑塊標(biāo)本區(qū)分為穩(wěn)定性斑塊組和不穩(wěn)定性斑塊組,通過(guò)高通量芯片篩選兩組差異的lncRNA和mRNA表達(dá)譜,進(jìn)而將篩選的差異基因進(jìn)行生物信息學(xué)分析,預(yù)測(cè)其可能的功能和生物學(xué)通路。(2)芯片結(jié)果驗(yàn)證:對(duì)芯片檢測(cè)出差異表達(dá)的lncRNA進(jìn)行real-time PCR篩選驗(yàn)證。經(jīng)過(guò)篩選,選擇lnc-RP11-159G9.5.1-2作為研究的目標(biāo)lncRNA,通過(guò)共表達(dá)分析和GO分析對(duì)lnc-RP11-159G9.5.1-2進(jìn)行生物信息學(xué)預(yù)測(cè)。(3)細(xì)胞分子研究:包裝干預(yù)細(xì)胞內(nèi)lnc-RP11-159G9.5.1-2上調(diào)和下調(diào)表達(dá)的慢病毒,通過(guò)慢病毒轉(zhuǎn)染,將血管平滑肌細(xì)胞分為lnc-RP11-159G9.5.1-2上調(diào)組、lnc-RP11-159G9.5.1-2上調(diào)陰性對(duì)照組,lnc-RP11-159G9.5.1-2下調(diào)組、lnc-RP11-159G9.5.1-2下調(diào)陰性對(duì)照組和空白對(duì)照組,通過(guò)cck8比色法法檢測(cè)lnc-RP11-159G9.5.1-2對(duì)平滑肌細(xì)胞的增殖功能的影響;通過(guò)流式細(xì)胞儀Annexin V FITC-PI檢測(cè)lnc-RP11-159G9.5.1-2對(duì)平滑肌細(xì)胞的凋亡功能的影響。結(jié)果(1)臨床樣本研究:2015年8月-2016年6月,共收集在長(zhǎng)海醫(yī)院血管外科中心行CEA手術(shù)治療患者標(biāo)本8例,通過(guò)高通量lncRNA基因芯片檢測(cè)頸動(dòng)脈穩(wěn)定性斑塊患者和不穩(wěn)定性斑塊患者斑塊組織中l(wèi)ncRNA的表達(dá),首次發(fā)現(xiàn)了差異表達(dá)的212種lncRNA,相較于穩(wěn)定組斑塊,不穩(wěn)定斑塊中115種lncRNA表達(dá)上調(diào),97種lncRNA表達(dá)下調(diào)。通過(guò)生物信息學(xué)分析,在GO分析和KEGG pathway分析中,差異表達(dá)的mRNA在調(diào)控血管大小、涉及凋亡的執(zhí)行階段的細(xì)胞組分解離、血小板聚集、細(xì)胞對(duì)血管內(nèi)皮生長(zhǎng)因子刺激的反應(yīng)、負(fù)向調(diào)節(jié)凋亡過(guò)程、血管內(nèi)皮生長(zhǎng)因子受體信號(hào)通路、正向調(diào)節(jié)內(nèi)皮細(xì)胞趨化性以及平滑肌細(xì)胞遷移等條目中均有富集,這些生物學(xué)功能涉及到頸動(dòng)脈斑塊的發(fā)生、發(fā)展和整個(gè)細(xì)胞周期過(guò)程。(2)芯片結(jié)果驗(yàn)證:芯片結(jié)果表明lnc-RP11-159G9.5.1-2在不穩(wěn)定斑塊的患者中高表達(dá),通過(guò)RT-PCR驗(yàn)證了lnc-RP11-159G9.5.1-2在不穩(wěn)定斑塊患者中相對(duì)表達(dá)量為4.666±1.741,在穩(wěn)定性斑塊患者中相對(duì)表達(dá)量為1.000±0.613,不穩(wěn)定斑塊lnc-RP11-159G9.5.1-2含量明顯高于穩(wěn)定性斑塊組(p0.0001)。(3)細(xì)胞分子研究:lnc-RP11-159G9.5.1-2可抑制血管平滑肌細(xì)胞的增殖,促進(jìn)血管平滑肌細(xì)胞的凋亡。結(jié)論應(yīng)用高通量lncRNA基因芯片方法獲得了頸動(dòng)脈狹窄穩(wěn)定性斑塊患者和不穩(wěn)定性斑塊患者差異的lncRNA表達(dá)譜,提示不穩(wěn)定斑塊頸動(dòng)脈狹窄患者中l(wèi)nc-RP11-159G9.5.1-2高表達(dá)。lnc-RP11-159G9.5.1-2通過(guò)抑制平滑肌細(xì)胞的增殖同時(shí)促進(jìn)平滑肌細(xì)胞的凋亡影響頸動(dòng)脈斑塊的穩(wěn)定性。提示不穩(wěn)定斑塊患者中l(wèi)nc-RP11-159G9.5.1-2的高表達(dá)抑制了斑塊纖維帽中平滑肌細(xì)胞的增殖同時(shí)促進(jìn)了凋亡,從而使平滑肌細(xì)胞減少,造成斑塊纖維帽變薄,易于破裂,最終導(dǎo)致斑塊不穩(wěn)定,更易于發(fā)生腦血管事件,從而影響了疾病的進(jìn)程和預(yù)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.

【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R543.4

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