【摘要】：研究背景原發(fā)性高血壓(primary hypertension)是臨床常見的一種慢性疾病,也是心腦血管病最主要的危險(xiǎn)因素,其主要并發(fā)癥如腦卒中、心肌梗死、心力衰竭及慢性腎病等,不僅致殘、致死率高,而且嚴(yán)重消耗醫(yī)療和社會資源,給家庭和社會造成沉重負(fù)擔(dān),對人類健康危害較大,其發(fā)病機(jī)制目前仍不明確。近年來,隨著我國人口老齡化、生活水平的改善、生活節(jié)奏加快等因素,原發(fā)性高血壓發(fā)病率持續(xù)升高,且年輕患者比例顯著增多,原發(fā)性高血壓的預(yù)防和治療引起了研究者們高度重視。血管損傷是高血壓疾病的基本病理改變之一,主要包括血管內(nèi)皮細(xì)胞損傷、平滑肌細(xì)胞增殖和血管重構(gòu)等病理過程。研究發(fā)現(xiàn),血管內(nèi)皮細(xì)胞損傷在高血壓的發(fā)生發(fā)展中具有重要的作用。例如：高血壓誘導(dǎo)的血管內(nèi)皮細(xì)胞功能失調(diào),破壞其NO與內(nèi)皮素的分泌穩(wěn)態(tài),誘導(dǎo)血管收縮加強(qiáng),從而增加了循環(huán)阻力,促進(jìn)高血壓的發(fā)展；在高血壓環(huán)境中,微血管內(nèi)皮細(xì)胞的凋亡率顯著升高,導(dǎo)致血液平行通路減少,提高了外周循環(huán)阻力,導(dǎo)致血壓升高。盡管越來越多的研究顯示,高血壓誘導(dǎo)的血管內(nèi)皮細(xì)胞損傷在高血壓發(fā)生發(fā)展中具有重要作用,但其分子機(jī)理目前尚不明確,仍有待進(jìn)一步研究。MicroRNA(miRNA)是一類長度為18-24核苷酸左右的非編碼小分子RNA,其進(jìn)化呈高度保守,主要通過與靶基因mRNA的3口非翻譯區(qū)(3□ untranslated region,3□-UTR)區(qū)完全或不完全結(jié)合,在轉(zhuǎn)錄后水平通過抑制蛋白質(zhì)翻譯或mRNA降解調(diào)控基因的表達(dá)。目前研究表明,高血壓患者外周血中miRNA表達(dá)譜發(fā)生了明顯改變,甚至具有高血壓早期診斷和預(yù)后判斷的臨床價(jià)值。此外,在高血壓引起的并發(fā)癥中, miRNA也發(fā)揮著重要的生物學(xué)功能。此外,多種miRNA分子也參與了血管內(nèi)皮細(xì)胞損傷。MiR-34a是一個多功能調(diào)控子,通過對相關(guān)靶基因的表達(dá)調(diào)控,參與細(xì)胞分裂、衰老、凋亡和增殖的調(diào)控。然而,1miRNA-34a在高血壓中的表達(dá)及作用機(jī)制仍不明確。在本研究中,我們首先檢測miR-34a在高血壓病患者外周血表達(dá)情況及其與高血壓病臨床病理特征之間的關(guān)系；然后,從體外實(shí)驗(yàn)方面進(jìn)行深入研究,探討miR-34a在高血壓內(nèi)皮細(xì)胞損傷中的作用及其機(jī)制,這可能為高血壓的臨床治療提供新的治療靶點(diǎn)。目的研究miR-34a在高血壓患者外周血中的表達(dá)及在高血壓誘導(dǎo)的血管內(nèi)皮細(xì)胞損傷中的分子調(diào)控機(jī)制。方法1.收集50例原發(fā)性高血壓患者外周血及28例正常健康人外周血,采用實(shí)時定量PCR檢測miR-34a的表達(dá)；2.將miR-34a抑制物(miR-34a inhibitor)以及其陰性對照(scramble control miRNA)分別轉(zhuǎn)入人臍靜脈內(nèi)皮細(xì)胞(HUVEC)中,轉(zhuǎn)染48h后進(jìn)行下一步檢測。3.分別采用CCK-8、Transwell及流式細(xì)胞術(shù)研究miR-34a分子對HUVECs的增殖、細(xì)胞周期、凋亡和遷移能力的影響。4.采用雙熒光素酶報(bào)告實(shí)驗(yàn)鑒定Tgif2是否為miR-34a靶基因。5.采用Western blot檢測下調(diào)miR-34a表達(dá)后,HUVECs中miR-34a潛在靶基因Tgif2的表達(dá)。6.采用SPSS16.0統(tǒng)計(jì)軟件進(jìn)行統(tǒng)計(jì)分析,所有計(jì)量數(shù)據(jù)均采用均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,分組比較使用成組t檢驗(yàn),以P0.05有統(tǒng)計(jì)學(xué)意義。結(jié)果：1.MiR-34a在高血壓患者外周血中的表達(dá)及臨床意義實(shí)時熒光定量PCR結(jié)果顯示,與正常對照組相比,高血壓患者外周血中miR-34a的表達(dá)明顯升高,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；在臨床病理特征分組比較中,高血壓3期患者外周血miR-34a表達(dá)明顯高于1期和2期患者(P0.05)；這一結(jié)果表明,高血壓患者外周血中miR-34a的表達(dá)上調(diào),且與高血壓進(jìn)展關(guān)系緊密。2. MiR-34a對HUVECs增殖能力的影響CCK-8實(shí)驗(yàn)結(jié)果顯示,下調(diào)miR-34a表達(dá)后,血管內(nèi)皮細(xì)胞]HUVECs的體外增殖能力明顯增強(qiáng),表明miR-34a具有抑制HUVECs的增殖的功能,其在高血壓患者外周血中的高表達(dá)可能抑制血管內(nèi)皮細(xì)胞的增殖。3. MiR-34a對HUVECs遷移能力的影響Transwell實(shí)驗(yàn)結(jié)果顯示,下調(diào)miR-34a表達(dá)后, HUVECs細(xì)胞遷移能力明顯升高,穿過小室膜的細(xì)胞數(shù)多于NC組(74.5±4.30 vs 48.5±6.3),差異有統(tǒng)計(jì)學(xué)意義(P0.05)；這一結(jié)果表明miR-34a能夠抑制HUVECs細(xì)胞遷移能力,在高血壓引起的血管內(nèi)皮損傷中,可能導(dǎo)致鄰近血管內(nèi)皮細(xì)胞向損傷處的遷移受抑,從而影響損傷血管內(nèi)皮細(xì)胞的修復(fù)。4. MiR-34a對HUVECs細(xì)胞周期的影響流式細(xì)胞術(shù)檢測miR-34a對HUVECs細(xì)胞周期的影響,結(jié)果顯示,與NC組相比,miR-34a inhibitor組的HUVECs出現(xiàn)了G1/S期轉(zhuǎn)換加快,表明過表達(dá)miR-34a能夠調(diào)控G1/S期轉(zhuǎn)換從而抑制HUVECs的增殖,從而抑制血管內(nèi)皮細(xì)胞的修復(fù)。5. MiR-34a對HUVECs凋亡的影響流式細(xì)胞儀檢測結(jié)果顯示,轉(zhuǎn)染miR-34a inhibitor后,人臍靜脈內(nèi)皮細(xì)胞的凋亡率明顯降低(P0.05),表明在高血壓發(fā)生發(fā)展過程中,降低miR-34a的表達(dá)能夠抑制HUVECs細(xì)胞凋亡,可能是高血壓誘導(dǎo)的血管內(nèi)皮損傷的作用機(jī)制之一。6. Tigf2為miR-34a的靶基因生物信息學(xué)分析顯示,miR-34a和Tgif2的3口-UTR區(qū)存在結(jié)合位點(diǎn),我們采用雙熒光素酶報(bào)告基因?qū)嶒?yàn)來確定Tgif2是否為miR-34a的直接靶基因。結(jié)果表明,與NC組相比,共轉(zhuǎn)染miR-34a mimics和Tigf2-wild type熒光素酶報(bào)告質(zhì)粒后,熒光值顯著下調(diào)(P0.05)；而Tigf2-mutant與miR-34a mimics共轉(zhuǎn)后,熒光值并無明顯差異(P0.05),表明miR-34a能與靶基因Tgif2 mRNA的3口-UTR種子區(qū)結(jié)合。免疫印跡結(jié)果顯示,下調(diào)1miR-34a表達(dá)水平后,HUVECs中Tigf2蛋白表達(dá)均出現(xiàn)明顯上升(P0.05)；提示miR-34a的生物學(xué)功能可能與Tigf2表達(dá)相關(guān)。結(jié)論：1. MiR-34a在高血壓患者外周血中表達(dá)顯著升高,且與高血壓臨床分級呈正相關(guān)；2. MiR-34a能夠通過下調(diào)Tgif2表達(dá)抑制HUVECs細(xì)胞的增殖和遷移能力,促進(jìn)其凋亡,從而促進(jìn)血管內(nèi)皮的損傷。
[Abstract]:Background Essential hypertension is a common chronic disease and the most important risk factor of cardiovascular and cerebrovascular diseases. Its main complications, such as stroke, myocardial infarction, heart failure and chronic kidney disease, not only cause disability and high mortality, but also seriously consume medical and social resources to create family and society. In recent years, with the aging of the population, the improvement of living standards, the quickening pace of life and other factors, the incidence of essential hypertension continues to rise, and the proportion of young patients significantly increased, the prevention and treatment of essential hypertension caused researchers. Vascular injury is one of the basic pathological changes in hypertension, including vascular endothelial cell injury, smooth muscle cell proliferation and vascular remodeling. It has been found that vascular endothelial cell injury plays an important role in the development of hypertension. It can disrupt the homeostasis of NO and ET secretion and induce vasoconstriction, which increases circulatory resistance and promotes the development of hypertension. In hypertensive environments, the apoptosis rate of microvascular endothelial cells increases significantly, leading to a decrease in blood parallel pathways, an increase in peripheral circulation resistance, and an increase in blood pressure. MicroRNA (microRNA) is a kind of non-coding small RNA with a length of 18-24 nucleotides, which is highly conserved and mainly through three ports of target gene mRNA. Complete or incomplete binding of the untranslated region (3_-UTR) region to regulate gene expression at the posttranscriptional level by inhibiting protein translation or mRNA degradation. Current studies have shown that the expression profile of microRNAs in the peripheral blood of hypertensive patients has changed significantly, and even has high clinical value in early diagnosis and prognosis of hypertension. MiR-34a is a multifunctional regulator involved in the regulation of cell division, senescence, apoptosis and proliferation by regulating the expression of related target genes. In this study, we first examined the expression of microRNAs in the peripheral blood of patients with hypertension and its relationship with the clinicopathological characteristics of hypertension; then, we conducted in-depth studies in vitro to explore the role and mechanism of microRNAs in the injury of hypertensive endothelial cells. Objective To investigate the expression of microRNA-34a in peripheral blood of patients with hypertension and its molecular regulation mechanism in vascular endothelial cell injury induced by hypertension. MicroRNA-34a inhibitor and scramble-controlled microRNA were transfected into human umbilical vein endothelial cells (HUVEC) for further detection 48 hours. CCK-8, Transwell and flow cytometry were used to study the proliferation, cell cycle, apoptosis of HUVECs. The expression of Tgif2 in HUVECs was detected by Western blot. 6. The expression of Tgif2 in HUVECs was analyzed by SPSS16.0 statistical software. All measurements were expressed by mean (+) standard deviation (x (+) s). Results: 1. The expression of MiR-34a in the peripheral blood of patients with hypertension and its clinical significance Real-time fluorescence quantitative PCR results showed that compared with the normal control group, the expression of MiR-34a in the peripheral blood of patients with hypertension was significantly higher, the difference was statistically significant (P 0.05). The expression of microRNA-34a in peripheral blood of patients with stage 3 hypertension was significantly higher than that of patients with stage 1 and 2 hypertension (P MiR-34a may inhibit the proliferation of vascular endothelial cells in hypertensive patients. 3. MiR-34a may affect the migration of HUVECs. Transwell experiment showed that HUVECs migration ability was inhibited by down-regulating the expression of MiR-34a. The number of cells passing through the ventricular membrane was significantly higher than that of NC group (74.5+4.30 vs 48.5+6.3), and the difference was statistically significant (P The effect of MiR-34a on the cell cycle of HUVECs was detected by flow cytometry. The results showed that G1/S phase transition of HUVECs in the inhibitor group was faster than that in the NC group, indicating that overexpression of MiR-34a could regulate G1/S phase transition and inhibit the proliferation of HUVECs, thereby inhibiting the proliferation of HUVECs. Effect of MiR-34a on apoptosis of HUVECs Flow cytometry showed that the apoptosis rate of HUVECs decreased significantly after transfection of MiR-34a inhibitor (P 0.05), suggesting that reducing the expression of MiR-34a could inhibit the apoptosis of HUVECs during the development of hypertension, which may be induced by hypertension. Tigf2 is a target gene for microRNAs-34a. Bioinformatics analysis showed that there were binding sites in the three-port-UTR region of microRNAs-34a and Tgif2. We used double luciferase reporter gene assay to determine whether Tgif2 is a direct target gene for microRNAs-34a. The fluorescence value of gf2-wild type luciferase reporter plasmid was significantly down-regulated (P 0.05), while the fluorescence value of Tigf2-mutant was not significantly different from that of Mimics (P 0.05), indicating that Mi-34a could bind to the three-port UTR seed region of target gene Tgif2 mRNA. Conclusion: 1. MiR-34a expression in peripheral blood of patients with hypertension was significantly increased, and was positively correlated with the clinical grade of hypertension; 2. MiR-34a could inhibit the proliferation and migration of HUVECs cells by down-regulating the expression of Tgif2 and promote their apoptosis. And promote vascular endothelial damage.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R544.1

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