同型半胱氨酸对血栓调节蛋白基因甲基化修饰及其与脑梗死发病的关系

发布时间：2018-01-07 21:36

本文关键词：同型半胱氨酸对血栓调节蛋白基因甲基化修饰及其与脑梗死发病的关系　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：背景与目的:脑血管病目前已经成为我国城乡居民死因之首,也是单病种致残率最高的疾病,脑梗死(Cerebral infarction,CI)是脑血管病中最常见者。脑梗死是环境因素和遗传因素共同作用导致的多因素疾病,而表观遗传学则是联系环境因素与遗传因素的桥梁。DNA甲基化是目前最常见,也是研究最透彻的表观遗传修饰形式,能够抑制基因表达,导致其生物学功能缺失,且这种表观遗传方式具有可逆性、可遗传性。高同型半胱氨酸血症(Hyperhomocysteinemia,HHcy)被认为是脑梗死的独立危险因素,其通过甲硫氨酸循环产生S-腺苷甲硫氨酸,后者作为DNA甲基化的唯一甲基供体来改变易感基因甲基化状态,进而参与多种疾病的发生发展。血栓调节蛋白(Thrombomodulin,TM)是由血管内皮细胞合成的膜糖蛋白,作为凝血酶受体,其具有抗凝、促纤溶、抗炎等功能,在预防脑梗死发病中起着至关重要的作用。已有研究显示冠心病患者外周血血栓调节蛋白基因启动子区发生异常高甲基化,但未见其与脑梗死的相关性研究。本研究通过探讨血栓调节蛋白基因启动子区异常甲基化改变在高同型半胱氨酸血症致脑梗死形成中的作用,从表观遗传学角度探讨同型半胱氨酸(Homocysteine,Hcy)作为脑梗死独立危险因素的致病机理,为防治脑梗死提供新的思路和理论依据。方法:采取病例-对照研究,入选120例脑梗死患者和80例对照,脑梗死患者来源于包头医学院第一附属医院神经内科2013年5月～2014年11月的住院患者,均符合全国第四届脑血管病会议修订的标准,并经CT或MRI检查证实有新发脑梗死病灶;对照为健康体检者和同期住院的良性阵发性位置性眩晕、偏头痛患者,病史及体格检查中无脑血管病证据和(或)头颅CT/MRI正常者。记录所有研究对象临床资料及高危因素。所有脑梗死患者均接受颅脑MRI检查,并记录梗死灶大小,采用美国国立卫生院神经功能缺损评分(National institutes of health stroke scale,NIHSS)评价神经功能缺损严重程度。所有研究对象抽取肘静脉血,提取基因组DNA,采用巢式甲基特异性PCR分析TM启动子区甲基化状态;提取血液总RNA,采用实时荧光定量PCR检测TMmRNA;采用ELISA法测定血浆可溶性血栓调节蛋白(solubleThrombomodulin,sTM)水平;荧光生化法检测血浆Hcy浓度。所有数据经SPSS17.0软件处理。结果:(1)脑梗死组与对照组相比,年龄、性别、甘油三酯之间的差异无统计学意义,胆固醇、吸烟及饮酒者所占比例、合并高血压及糖尿病的比例之间的差异有统计学意义。(2)TM基因启动子区甲基化状态包括非甲基化、部分甲基化和完全甲基化三种。脑梗死组TM基因启动子区甲基化率(74.2%)明显高于对照组(47.5%),差异具有统计学意义(X~2=14.724,p=0.00);头颅MRI弥散相测得脑梗死组梗死灶直径为3.32±2.49cm,依据其大小将脑梗死患者分为大面积脑梗死组、中等脑梗死组与腔隙性脑梗死组三个亚组,其甲基化率分别为90.3%,76.2%,61.7%,随着梗死病灶扩大,TM基因甲基化率有逐渐增加趋势,但仅大面积脑梗死组与腔隙性脑梗死组差异有统计学意义(X~2 =7.777,p=0.005),大面积脑梗死组与中等面积脑梗死组、中等面积脑梗死组与腔隙性脑梗死组间比较差异无统计学意义;脑梗死组入院后24小时内NIHSS评分为6.55±4.25分,与TM基因启动子区甲基化状态进行spearman秩相关分析,二者呈正相关(r=0.462,p=0.00)。(3)脑梗死患者血浆Hcy浓度(14.31±4.61μmol/L)明显高于对照组(10.25±2.97μmol/L),差异有统计学意义(t=7.566,p=0.00)。在脑梗死组,TM基因启动子区甲基化程度与血浆Hcy浓度呈高度正相关(r=0.701,p=0.00)。(4)将脑梗死患者TM基因甲基化状态与各种脑血管病高危因素进行二元logistic相关分析,发现年龄(OR=2.097,p=0.031)、吸烟(OR=3.091,p=0.027)是脑梗死患者TM基因甲基化状态的影响因素,而性别、饮酒、血糖与LDL不是脑梗死患者TM基因甲基化状态的影响因素。(5)脑梗死患者TM mRNA表达水平明显低于对照组,为对照组的27%,脑梗死组按照TM基因启动子区甲基化状态分组,非甲基化组、部分甲基化组、完全甲基化组TM mRNA表达水平逐渐降低,分别为对照组的64%、25%、12%,TM mRNA表达水平与其启动子区甲基化状态呈高度负相关(r=-0.711,p=0.00)。(6)给予50例脑梗死合并高同型半胱氨酸血症患者口服维生素B6、甲钴胺、叶酸治疗12周,治疗后血浆Hcy浓度(12.36±2.80μmol/L)较治疗前(19.02±2.44μmol/L)明显降低,差异有统计学意山东大学博士学位论文义(t=16.314,p=0.00);治疗后TM基因启动子区完全甲基化率(42%)较治疗前(54%)有所下降,但差异无统计学意义(X~2=1.442,p=0.230);治疗后TM基因mRNA表达(0.2514±0.1295)较治疗前(0.2149±0.1170)升高,且差异有统计学意义(t=3.205,p=0.02)。(7)脑梗死患者血浆sTM浓度(4.33±0.84ng/ml)明显高于对照组(2.81±0.38ng/ml),差异有统计学意义(t=17.268,p=0.00)。脑梗死患者梗死灶直径为3.32:±2.49cm,与其血浆sTM浓度呈正相关(r=0.611,p=0.00);脑梗死患者入院后NIHSS评分6.55±4.25分,与其血浆sTM浓度呈正相关(r=0.544,p=0.00)。结论:(1)脑梗死患者TM基因启动子区甲基化率明显高于对照组,且其与脑梗死患者血浆Hcy浓度呈高度正相关,与脑梗死灶大小、入院后NIHSS评分呈正相关;与TM mRNA表达水平呈负相关;提示Hcy可能通过诱导TM基因启动子区高甲基化而使其mRNA表达减少,降低TM的抗凝、促纤溶、抗炎等功能,进而参与脑梗死的发生、发展。(2)年龄与吸烟是脑梗死患者TM基因甲基化状态的影响因素,而性别、饮酒、血糖与LDL不是脑梗死患者TM基因甲基化状态的影响因素。(3)口服叶酸、维生素B12与维生素B612周后能够明显降低脑梗死患者血浆Hcy浓度,但逆转其引起的外周血TM基因高甲基化状态可能需要更长时间。(4)脑梗死患者血浆sTM水平升高,与其脑梗死灶大小、入院后NIHSS评分呈正相关,但其sTM水平增高并非TM基因表达增多所致,而是因脑梗死患者血管内皮受损释放入血所致,可作为反映血管内皮细胞损伤程度的指标。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3

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