發(fā)布時(shí)間：2018-01-07 21:36

  本文關(guān)鍵詞：同型半胱氨酸對(duì)血栓調(diào)節(jié)蛋白基因甲基化修飾及其與腦梗死發(fā)病的關(guān)系　出處：《山東大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 血栓調(diào)節(jié)蛋白 同型半胱氨酸 甲基化 腦梗死

【摘要】：背景與目的:腦血管病目前已經(jīng)成為我國城鄉(xiāng)居民死因之首,也是單病種致殘率最高的疾病,腦梗死(Cerebral infarction,CI)是腦血管病中最常見者。腦梗死是環(huán)境因素和遺傳因素共同作用導(dǎo)致的多因素疾病,而表觀遺傳學(xué)則是聯(lián)系環(huán)境因素與遺傳因素的橋梁。DNA甲基化是目前最常見,也是研究最透徹的表觀遺傳修飾形式,能夠抑制基因表達(dá),導(dǎo)致其生物學(xué)功能缺失,且這種表觀遺傳方式具有可逆性、可遺傳性。高同型半胱氨酸血癥(Hyperhomocysteinemia,HHcy)被認(rèn)為是腦梗死的獨(dú)立危險(xiǎn)因素,其通過甲硫氨酸循環(huán)產(chǎn)生S-腺苷甲硫氨酸,后者作為DNA甲基化的唯一甲基供體來改變易感基因甲基化狀態(tài),進(jìn)而參與多種疾病的發(fā)生發(fā)展。血栓調(diào)節(jié)蛋白(Thrombomodulin,TM)是由血管內(nèi)皮細(xì)胞合成的膜糖蛋白,作為凝血酶受體,其具有抗凝、促纖溶、抗炎等功能,在預(yù)防腦梗死發(fā)病中起著至關(guān)重要的作用。已有研究顯示冠心病患者外周血血栓調(diào)節(jié)蛋白基因啟動(dòng)子區(qū)發(fā)生異常高甲基化,但未見其與腦梗死的相關(guān)性研究。本研究通過探討血栓調(diào)節(jié)蛋白基因啟動(dòng)子區(qū)異常甲基化改變?cè)诟咄�型半胱氨酸血癥致腦梗死形成中的作用,從表觀遺傳學(xué)角度探討同型半胱氨酸(Homocysteine,Hcy)作為腦梗死獨(dú)立危險(xiǎn)因素的致病機(jī)理,為防治腦梗死提供新的思路和理論依據(jù)。方法:采取病例-對(duì)照研究,入選120例腦梗死患者和80例對(duì)照,腦梗死患者來源于包頭醫(yī)學(xué)院第一附屬醫(yī)院神經(jīng)內(nèi)科2013年5月～2014年11月的住院患者,均符合全國第四屆腦血管病會(huì)議修訂的標(biāo)準(zhǔn),并經(jīng)CT或MRI檢查證實(shí)有新發(fā)腦梗死病灶;對(duì)照為健康體檢者和同期住院的良性陣發(fā)性位置性眩暈、偏頭痛患者,病史及體格檢查中無腦血管病證據(jù)和(或)頭顱CT/MRI正常者。記錄所有研究對(duì)象臨床資料及高危因素。所有腦梗死患者均接受顱腦MRI檢查,并記錄梗死灶大小,采用美國國立衛(wèi)生院神經(jīng)功能缺損評(píng)分(National institutes of health stroke scale,NIHSS)評(píng)價(jià)神經(jīng)功能缺損嚴(yán)重程度。所有研究對(duì)象抽取肘靜脈血,提取基因組DNA,采用巢式甲基特異性PCR分析TM啟動(dòng)子區(qū)甲基化狀態(tài);提取血液總RNA,采用實(shí)時(shí)熒光定量PCR檢測TMmRNA;采用ELISA法測定血漿可溶性血栓調(diào)節(jié)蛋白(solubleThrombomodulin,sTM)水平;熒光生化法檢測血漿Hcy濃度。所有數(shù)據(jù)經(jīng)SPSS17.0軟件處理。結(jié)果:(1)腦梗死組與對(duì)照組相比,年齡、性別、甘油三酯之間的差異無統(tǒng)計(jì)學(xué)意義,膽固醇、吸煙及飲酒者所占比例、合并高血壓及糖尿病的比例之間的差異有統(tǒng)計(jì)學(xué)意義。(2)TM基因啟動(dòng)子區(qū)甲基化狀態(tài)包括非甲基化、部分甲基化和完全甲基化三種。腦梗死組TM基因啟動(dòng)子區(qū)甲基化率(74.2%)明顯高于對(duì)照組(47.5%),差異具有統(tǒng)計(jì)學(xué)意義(X~2=14.724,p=0.00);頭顱MRI彌散相測得腦梗死組梗死灶直徑為3.32±2.49cm,依據(jù)其大小將腦梗死患者分為大面積腦梗死組、中等腦梗死組與腔隙性腦梗死組三個(gè)亞組,其甲基化率分別為90.3%,76.2%,61.7%,隨著梗死病灶擴(kuò)大,TM基因甲基化率有逐漸增加趨勢,但僅大面積腦梗死組與腔隙性腦梗死組差異有統(tǒng)計(jì)學(xué)意義(X~2 =7.777,p=0.005),大面積腦梗死組與中等面積腦梗死組、中等面積腦梗死組與腔隙性腦梗死組間比較差異無統(tǒng)計(jì)學(xué)意義;腦梗死組入院后24小時(shí)內(nèi)NIHSS評(píng)分為6.55±4.25分,與TM基因啟動(dòng)子區(qū)甲基化狀態(tài)進(jìn)行spearman秩相關(guān)分析,二者呈正相關(guān)(r=0.462,p=0.00)。(3)腦梗死患者血漿Hcy濃度(14.31±4.61μmol/L)明顯高于對(duì)照組(10.25±2.97μmol/L),差異有統(tǒng)計(jì)學(xué)意義(t=7.566,p=0.00)。在腦梗死組,TM基因啟動(dòng)子區(qū)甲基化程度與血漿Hcy濃度呈高度正相關(guān)(r=0.701,p=0.00)。(4)將腦梗死患者TM基因甲基化狀態(tài)與各種腦血管病高危因素進(jìn)行二元logistic相關(guān)分析,發(fā)現(xiàn)年齡(OR=2.097,p=0.031)、吸煙(OR=3.091,p=0.027)是腦梗死患者TM基因甲基化狀態(tài)的影響因素,而性別、飲酒、血糖與LDL不是腦梗死患者TM基因甲基化狀態(tài)的影響因素。(5)腦梗死患者TM mRNA表達(dá)水平明顯低于對(duì)照組,為對(duì)照組的27%,腦梗死組按照TM基因啟動(dòng)子區(qū)甲基化狀態(tài)分組,非甲基化組、部分甲基化組、完全甲基化組TM mRNA表達(dá)水平逐漸降低,分別為對(duì)照組的64%、25%、12%,TM mRNA表達(dá)水平與其啟動(dòng)子區(qū)甲基化狀態(tài)呈高度負(fù)相關(guān)(r=-0.711,p=0.00)。(6)給予50例腦梗死合并高同型半胱氨酸血癥患者口服維生素B6、甲鈷胺、葉酸治療12周,治療后血漿Hcy濃度(12.36±2.80μmol/L)較治療前(19.02±2.44μmol/L)明顯降低,差異有統(tǒng)計(jì)學(xué)意山東大學(xué)博士學(xué)位論文義(t=16.314,p=0.00);治療后TM基因啟動(dòng)子區(qū)完全甲基化率(42%)較治療前(54%)有所下降,但差異無統(tǒng)計(jì)學(xué)意義(X~2=1.442,p=0.230);治療后TM基因mRNA表達(dá)(0.2514±0.1295)較治療前(0.2149±0.1170)升高,且差異有統(tǒng)計(jì)學(xué)意義(t=3.205,p=0.02)。(7)腦梗死患者血漿sTM濃度(4.33±0.84ng/ml)明顯高于對(duì)照組(2.81±0.38ng/ml),差異有統(tǒng)計(jì)學(xué)意義(t=17.268,p=0.00)。腦梗死患者梗死灶直徑為3.32:±2.49cm,與其血漿sTM濃度呈正相關(guān)(r=0.611,p=0.00);腦梗死患者入院后NIHSS評(píng)分6.55±4.25分,與其血漿sTM濃度呈正相關(guān)(r=0.544,p=0.00)。結(jié)論:(1)腦梗死患者TM基因啟動(dòng)子區(qū)甲基化率明顯高于對(duì)照組,且其與腦梗死患者血漿Hcy濃度呈高度正相關(guān),與腦梗死灶大小、入院后NIHSS評(píng)分呈正相關(guān);與TM mRNA表達(dá)水平呈負(fù)相關(guān);提示Hcy可能通過誘導(dǎo)TM基因啟動(dòng)子區(qū)高甲基化而使其mRNA表達(dá)減少,降低TM的抗凝、促纖溶、抗炎等功能,進(jìn)而參與腦梗死的發(fā)生、發(fā)展。(2)年齡與吸煙是腦梗死患者TM基因甲基化狀態(tài)的影響因素,而性別、飲酒、血糖與LDL不是腦梗死患者TM基因甲基化狀態(tài)的影響因素。(3)口服葉酸、維生素B12與維生素B612周后能夠明顯降低腦梗死患者血漿Hcy濃度,但逆轉(zhuǎn)其引起的外周血TM基因高甲基化狀態(tài)可能需要更長時(shí)間。(4)腦梗死患者血漿sTM水平升高,與其腦梗死灶大小、入院后NIHSS評(píng)分呈正相關(guān),但其sTM水平增高并非TM基因表達(dá)增多所致,而是因腦梗死患者血管內(nèi)皮受損釋放入血所致,可作為反映血管內(nèi)皮細(xì)胞損傷程度的指標(biāo)。
[Abstract]:Background and purpose: cerebrovascular disease has become the cause of urban and rural residents in China for the first time, is also a single disease with high incidence of disability disease, cerebral infarction (Cerebral infarction CI) is the most common cerebrovascular disease. Cerebral infarction is a multifactorial disease caused by genetic and environmental factors, and epigenetics is environmental and genetic factors of bridge.DNA methylation is the most common form of epigenetic modifications is the most thorough, can inhibit the gene expression, biological function and lead to the lack of this epigenetic type is reversible, can be inherited. Hyperhomocysteinemia (Hyperhomocysteinemia, HHcy) is that is an independent risk factor for cerebral infarction, through the methionine cycle S- S-adenosylmethionine, only the latter as the methyl DNA methylation donor to change susceptibility gene methylation, The occurrence and development and are involved in many diseases. Thrombomodulin (Thrombomodulin, TM) is a synthesis of vascular endothelial cell membrane glycoprotein, as thrombin receptor, which has anticoagulant, fibrinolysis, anti-inflammatory and other functions, in the prevention of cerebral infarction plays a vital role. Studies have shown that peripheral blood thrombosis in patients with coronary heart disease control protein gene promoter hypermethylation is abnormal, but no correlation with cerebral infarction. The study investigated thrombomodulin gene promoter hypermethylation in hyperhomocysteinemia induced by cerebral infarction in the formation, from the perspective of epigenetics on homocysteine (Homocysteine, Hcy) as a disease the mechanism of independent risk factors of cerebral infarction, and provide new ideas and theoretical basis for the prevention and treatment of cerebral infarction. Methods: a case-control study in 120 cases of cerebral infarction Patients and 80 control cases, cerebral infarction patients from the First Affiliated Hospital of Baotou Medical College Department of Neurology in May 2013 ~ November 2014 in hospitalized patients, are consistent with the revision of the fourth national Cerebrovascular Disease Conference Standard, and the CT or MRI examination confirmed cerebral infarction lesions; control in healthy persons and the same period of benign paroxysmal positional vertigo, migraine, history and physical examination without evidence of cerebrovascular disease (or head) and normal CT/MRI. Record the clinical data of all the subjects and the risk factors of cerebral infarction. All patients underwent brain MRI examination, and record the infarct size, the National Institutes of Health Stroke (National Institutes of Health Stroke score scale, NIHSS) to evaluate the neurological severity. All subjects from the elbow vein blood, extract genomic DNA by nested methylation specific PCR analysis of TM promoter methylation; RNA extraction in blood, detected by real-time fluorescent quantitative PCR TMmRNA; Determination of plasma soluble thrombomodulin by ELISA (solubleThrombomodulin, sTM); to detect the concentration of plasma Hcy fluorescence biochemical method. All data with SPSS17.0 software. Results: (1) the cerebral infarction group compared with the control group in age, gender, no significant difference between cholesterol and triglyceride, smoking drinkers proportion, the difference was statistically significant between hypertension and diabetes. The proportion (2) of TM gene promoter methylation status including non methylation, partial methylation and full methylation of three cerebral infarction group. TM gene promoter methylation rate (74.2%) was significantly higher than the control group (47.5%), the difference was statistically significant (X~2=14.724, p=0.00); head MRI dispersed phase measured cerebral infarction infarction diameter is 3 .32 + 2.49cm, according to the size of the large area cerebral infarction were divided into cerebral infarction group, middle cerebral infarction group and lacunar infarction group three subgroups, the methylation rates were 90.3%, 76.2%, 61.7%, with the expansion of the cerebral infarction, the TM gene methylation rate increased gradually, but is only significant in large area the cerebral infarction group and lacunar infarction group (X~2 =7.777, p=0.005), a large area of cerebral infarction group and moderate infarct group, middle size cerebral infarction group and lacunar infarction group was no significant difference between groups; cerebral infarction admitted to hospital within 24 hours after the NIHSS score was 6.55 + 4.25, Spearman rank correlation analysis and TM gene promoter methylation, two were positively correlated (r=0.462, p=0.00). (3) the concentration of plasma Hcy in patients with cerebral infarction (14.31 + 4.61 mol/L) was significantly higher than the control group (10.25 + 2.97 u mol/L), the difference was statistically significant (t= 7.566, p=0.00). In the cerebral infarction group, TM gene promoter methylation level was positively correlated with the plasma concentration of Hcy (r=0.701, p=0.00). (4) the state and various factors of cerebrovascular disease in high-risk TM patients with cerebral infarction were two yuan logistic gene methylation analysis, found that age (OR=2.097, p=0.031) (OR=3.091, p=0.027), smoking is a factor, effect of cerebral infarction in patients with TM gene methylation status and gender, alcohol consumption, influencing factors of blood glucose and LDL in patients with cerebral infarction than the methylation status of TM gene in patients with cerebral infarction (5). TM mRNA expression level was significantly lower than the control group, the control group 27%, cerebral infarction group according to TM gene the methylation status of the promoter region into non methylation group, partial methylation group, total methylation group TM mRNA expression level decreased gradually, the control group respectively 64%, 25%, 12%, TM mRNA expression level was highly negative with promoter hypermethylation Related (r=-0.711, p=0.00). (6) to 50 cases of cerebral infarction with hyperhomocysteinemia in patients with oral vitamin B6, methylcobalamin, folic acid for 12 weeks, the plasma concentration of Hcy after treatment (12.36 + 2.80 mol/L) than before treatment (19.02 + 2.44 mol/L) significantly decreased, the difference had statistical meaning of Shandong University Ph.D. the meaning of (t=16.314, p=0.00); after the treatment of TM gene promoter methylation rate (42%) than before treatment (54%) decreased, but the difference was not statistically significant (X~2=1.442, p=0.230); the expression of TM gene after mRNA treatment (0.2514 + 0.1295) than before treatment (0.2149 + 0.1170) has increased the difference was significant (t=3.205, p=0.02). (7) the plasma sTM concentration in patients with cerebral infarction (4.33 + 0.84ng/ml) was significantly higher than the control group (2.81 + 0.38ng/ml), the difference was statistically significant (t=17.268, p=0.00). Cerebral infarction lesions with diameter of 3.32: + 2.49cm, and plasma concentration of sTM Related (r=0.611, p=0.00); patients after cerebral infarction NIHSS score 6.55 + 4.25, and the concentration of plasma sTM was positively correlated (r=0.544, p=0.00). Conclusion: (1) cerebral infarction in patients with TM gene promoter methylation rate was significantly higher than the control group, and the concentration of plasma Hcy in patients with cerebral infarction was highly related to the size. With cerebral infarction after admission, NIHSS score was negatively correlated with the level of TM; mRNA expression; it was suggested that Hcy could induce TM gene promoter hypermethylation and the decreased expression of mRNA, TM reduced the anticoagulant, fibrinolysis, anti-inflammatory and other functions, and then participate in the occurrence of cerebral infarction (2). Age and smoking are the factors, effects of cerebral infarction in patients with TM gene methylation status and gender, alcohol consumption, influencing factors of blood glucose and LDL in patients with cerebral infarction than the methylation status of TM gene. (3) oral folic acid, vitamin B12 and vitamin B612 can significantly reduce the week after The concentration of plasma Hcy in patients with cerebral infarction, but the reversal caused by peripheral blood TM gene hypermethylation may take longer. (4) elevated plasma sTM levels in patients with cerebral infarction, and cerebral infarct size after admission, the NIHSS score was positively correlated, but the level of sTM increased TM gene expression is not caused by increased, but due to vascular patients endothelial damage caused by cerebral infarction is released into the blood, can reflect the damage degree of endothelial cells.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R743.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 徐營營;周曉艷;謝兆宏;許順良;于君;畢建忠;;雌激素受體α基因甲基化與缺血性卒中的相關(guān)性研究[J];中國動(dòng)脈硬化雜志;2015年10期

2 蔣承建;郭航遠(yuǎn);唐偉良;池菊芳;翟小亞;孟立平;郭艷;;同型半胱氨酸對(duì)內(nèi)皮祖細(xì)胞生成eNOS、p-eNOS和NO的影響及黃酒的改善作用[J];中國動(dòng)脈硬化雜志;2015年05期

3 陳大鵬;賈紹斌;叢廣志;;Hhcy誘導(dǎo)基因組低甲基化作用的研究[J];寧夏醫(yī)學(xué)雜志;2014年02期

4 劉靜;賈紹斌;侯建軍;張政軍;;瑞舒伐他汀在高同型半胱氨酸血癥誘導(dǎo)大鼠動(dòng)脈粥樣硬化中對(duì)基因組甲基化影響[J];臨床心血管病雜志;2013年04期

5 楊安寧;王菊;楊曉玲;馬長劍;孫煒煒;馬勝超;王磊;徐華;姜怡鄧;;同型半胱氨酸引起平滑肌細(xì)胞LDLR啟動(dòng)子區(qū)DNA甲基化改變的位點(diǎn)分析[J];中國藥理學(xué)通報(bào);2012年11期

6 叢廣志;賈紹斌;羅彩琴;;葉酸對(duì)高同型半胱氨酸血癥大鼠主動(dòng)脈基因組總甲基化水平影響研究[J];中國現(xiàn)代醫(yī)學(xué)雜志;2012年17期

7 楊志甫;王麗珍;孟祥君;張茂林;席富強(qiáng);;高同型半胱氨酸血癥對(duì)雌激素受體α基因甲基化修飾的影響及其與腦梗死發(fā)病的關(guān)系[J];山東醫(yī)藥;2011年41期

8 張偉麗;祝立新;孫凱;張春玲;惠汝太;汪道文;廖玉華;萬魯虹;;高同型半胱氨酸水平與腦卒中患者心腦血管事件再發(fā)風(fēng)險(xiǎn)的關(guān)系[J];臨床內(nèi)科雜志;2009年10期

9 黃丹丹;王樹人;;同型半胱氨酸致巨噬細(xì)胞TNFSF4基因啟動(dòng)子去甲基化并增加其mRNA表達(dá)[J];第三軍醫(yī)大學(xué)學(xué)報(bào);2008年07期

10 劉軍須;張敬各;;同型半胱氨酸經(jīng)甲基化修飾下調(diào)去卵巢大鼠主動(dòng)脈雌激素受體α基因的表達(dá)[J];基礎(chǔ)醫(yī)學(xué)與臨床;2008年03期

，

本文編號(hào)：1394320