【作者】 肖健齐；

【导师】 王运杰；

【作者基本信息】 中国医科大学 ， 神经外科， 2010， 博士

【摘要】 目的脑出血(intracerebral hemorrhage, ICH)是指源于脑实质内血管的非创伤性自发出血。出血也可破入脑室或蛛网膜下腔。ICH占所有卒中的10%—15%。脑出血可以发生在脑实质的任何部位,可以单发,也可为多发。原发性ICH通常是由于长期高血压或淀粉样脑血管病(cerebral amyloid angiopathy, CAA)或脑动脉粥样硬化等作用下发生病理改变的小血管或小动脉自发破裂造成的。脑出血是受环境因素和遗传因素共同影响的多因素疾病,流行病学研究显示高血压、高脂血症、高龄、过量饮酒、吸烟、身体质量指数、糖尿病是ICH的危险因素。近年来,随着脑出血家族聚集现象的发现,遗传因素在脑出血发病中的作用引起了人们的关注,成为研究的热点。进入21世纪越来越多的研究发现NFAT家族可作为细胞内多信号转导通路的基础因子,可以调节T细胞、B细胞内很多因子和IL-2、IL-3、IL-4、IL-5、IL-6、粒细胞-巨噬细胞集落刺激因子、肿瘤坏死因子-α(tumor necrosis factor, TNF-α)和环氧化酶-2(cyclooxygenase-2, COX-2、Apob、nurr77等多种细胞因子的表达水平。由于TNF-α基因与原发性高血压和血脂异常及动脉粥样硬化密切相关,COX-2与动脉粥样硬化密切相关,Apob、IL-6、nur77、SREBP-1与血脂异常密切相关,T细胞与血管内膜炎性反应密切相关,而NFATC1参与调解TNF-α的活化,在TNF-α启动因子中包含转录因子NFATC1潜在的结合位,NFATC1对TNF-α基因活化起重要作用,NFATC1参与调解COX-2的表达,NFATC1的调控与COX-2的表达呈正相关,NFATC1基因exon-2上存在转录因子SREBP-1的绑定位点,直接参与对SREBP-1因子表达的负调控,NFATC1还参与调解nur77的表达,NFATC1通过调控T细胞内诸多因子的表达使在正常动脉内膜未见或罕见T细胞在病变区域广泛浸润到血管内膜,产生炎性反应。因为原发性高血压是脑出血的重要的独立预测因素,血脂异常及脑动脉粥样硬化是脑出血的重要病理基础,NFATC1参与调控与高血压、动脉硬化、血脂异常密切相关的因子,NFATC1通过调控T细胞内多种因子参与病变区域血管内膜的炎性反应,所以我们有理由相信转录因子NFATC1是与脑出血性卒中密切相关的基因。目前转录因子NFATC1在脑出血中的表达及作用机制尚不明确,尤其是脑出血与NFATC1基因多态性是否存在联系在国内外尚没有研究,这需要我们进行大量工作以明确NFATC1基因多态性与脑出血的关系,从分子水平探讨脑出血的发病机制,若此相关性存在,在人群中进行该基因调查,可及早发现高危脑出血人群,并实施个性化治疗,从而更有效的预防和控制脑出血的发生。将给临床预防和治疗脑出血的工作带来新的希望。方法采用病例-对照研究方法,病例组选取中国北方脑出血患者230例,男144例、女86例,年龄在33-85岁间,均为汉族,彼此无亲缘关系,对照组选取性别、年龄、民族与病例组相匹配的健康个体233例,男146例、女87例本研究采用问卷调查及查体等方式,记录被研究者的基本情况(性别、年龄、BMI、收缩压、舒张压、嗜烟酒史等),采用日立7600全自动生化分析仪测定脑出血患者和正常人群血清的总胆固醇(total cholesterol, CHO)、甘油三脂(triacylglycerol, TG)、低密度脂蛋白(low density lipoprotein, LDL)及高密度脂蛋(high density lipoprotein, HDL)。载脂蛋白-a(Apo-a)、载脂蛋白-b(Apo-b)值,采用双脱氧末端终止测序法研究了NFATC1第2外显子上的功能性SNPS,聚合酶链反应方法研究了第7内含子可变数量串联重复多态性(VNTR)、多聚酶链式反应和限制性片段长度多态性(PCR-RFLP)方法研究第9号外显子上的多态性位点rs754093(Cys751Gly)与脑出血的关系,此结果再经双脱氧末端终止测序法进一步确认。用拟合优度卡方检验验证每个位点基因型在抽样群体中的分布是否符合H-W平衡,应用统计分析软件包(Statistical Packages for Social Sciences, SPSS13.0)和EXCEL建立数据库,需要统计的计量资料均进行正态性检验,正态分布的各统计资料均以均数±标准差表示,偏态分布先进行对数转换,再行比较。采用t检验方法分析病例组与对照组中血脂等计量资料的差异,χ2检验方法分析病例组与对照组人群基本背景情况计数资料的差异,四格表和R×C列表χ2检验方法分析病例组与对照组中等位基因和基因型的频数分布差异,单因素方差分析方法分析病例组各基因型间血脂关系的差异。P<0.05为统计学显著性差异。采用Logistic回归分析判断脑出血各危险因素作用的大小,分别计算各自的比值比(OR)及其95%可信区间(CI)。以OR的95%CI不包含1为统计学显著性差异。结果1、病例组与对照组在性别(Gender)、年龄(Age)、体质质量指数(BMI)构成上无统计学差异(P>0.05)。病例组中患高血压病(Hypertension)、糖尿病(Diabetesmellitus)、高脂血症(hyperlipidemia)及嗜烟(Smoking)嗜酒(drinking)者与对照组相比,显著增高(P<0.05)。病例组的甘油三脂(TG)、载脂蛋白a(Apoa)与对照组相比,差异无统计学意义(P>0.05)；病例组高密度脂蛋白(HDL)、低密度脂蛋白(LDL)、载脂蛋白b (Apob)、收缩压(SBP)、舒张压(DBP)、均显著升高,而总胆固醇(CHO)显著降低,与对照组相比有显著性差异(P<0.05)。2、所有研究对象的每个多态性基因型在抽样群体中的分布均符合H-W平衡,表示所选取的样本具有人群代表性适合做遗传学分析。3、经双脱氧末端终止测序法鉴定第2外显子rs1051978、rs62096875、rs12605457、rs1063669、rs61731547各位点SNP基因型,rs1051978为A／C二态,A／A基因型频率病例组低于对照组,A／C基因型频率病例组高于对照组,基因型频率比较χ2=0.349,P=0.555,；等位基因频率比较χ2=0.35,P=0.661。经卡方检验病例组和对照组此基因型分布无显著性差异。rs62096875为G／G纯合子,rs12605457为C／C纯合子、rs1063669为G／G纯合子、rs61731547为C／C纯合子,没有发现新的突变位点。4、第7内含子可变数量串联重复(variable number tandem repeat VNTR) L/L、S／S基因型频率病例组均低于对照组,而L／S基因型频率高于对照组,基因型频率比较χ2=0.957,P=0.620,等位基因频率比较χ2=0.12,P=0.727。经卡方检验病例组和对照组基因型分布无显著性差异。5、NFATC1第9外显子SNP rs754093病例组与对照组相比,T／T、T／G基因型频率均低于对照组,而G／G基因型频率明显高于对照组,基因型频率比较χ2=6.343,df=2,P=0.042；T／G等位基因频率比较χ2=6.39,df=1,P=0.011,OR=1.411,95%CI=1.080-1.843。经卡方检验病例组和对照组此基因型分布存在显著性差异。6、病例组rs754093 T／G各基因型与各项血脂、血压指标的关系,经方差分析(ANOVA)发现各基因型之间CHO、LDL、Apob和收缩压存在显著性差异。组间两两比较显示,GG基因型的CHO、LDL、Apob明显高于T／T和T／G基因型。GG基因型的CHO与T／T和T／G基因型比较F=10.423,P=0.000存在显著性差异,GG基因型的LDL与T／T和T／G基因型比较F=23.398,P=0.000存在显著性差异,GG基因型的Apob与T／T和T／G基因型比较F=32.176,P=0.000存在显著性差异,GG基因型的收缩压高于T／G基因型且明显高于T／T基因型,GG基因型的收缩压与T／T基因型比较F=2.129,P=0.042存在显著性差异。rs754093 T／G各基因型间糖尿病,嗜烟,嗜酒,BMI等情况无显著性差异。7、通过应用多元逐步Logistic回归分析,得出rs754093 GG基因型是脑出血的独立的危险因素之一(OR=3.902,95%CI 1.735～8.755,P=0.001)。此外,与脑出血相关的其他危险因素有LDL、Apob、收缩压升高。结论1、高血压病、糖尿病、高脂血症及嗜烟、嗜酒与脑出血密切相关,高密度脂蛋白、低密度脂蛋白、载脂蛋白b、收缩压、舒张压、总胆固醇与脑出血密切相关。甘油三脂、载脂蛋白a与脑出血无显著相关性。2、第2外显子上的功能性SNPS rs1051978、rs62096875、rs12605457、rs1063669、rs61731547与中国辽宁地区汉族人群脑出血没有显著的相关性。3、第7内含子可变数量串联重复(VNTR) L/S基因型与中国辽宁地区汉族人群脑出血的发病无相关性。4、NFATC1第9外显子上的rs754093 G／G基因型是中国辽宁地区汉族人群脑出血独立危险因素,其风险性主要来源于G等位基因。5、NFATC1基因是与中国辽宁地区汉族人群脑出血发病相关的易感性基因,NFATC1基因的SNP rs754093 T／G等位基因可能是脑出血易感突变位点,G等位基因与脑出血的风险增加有关.6、NFATC1 rs754093 G/G基因型与脑出血发病危险因素CHO LDL、Apob、收缩压升高密切相关。7、LDL、Apob、收缩压升高也是脑出血的独立危险因素。更多还原

【Abstract】 ObjectiveCerebral hemorrhage refers to the non-invasive spontaneous bleeding Of the blood vessels within the brain parenchyma. Bleeding can also be broken into the brain ventricles or subarachnoid space.ICH accounts for 10%-15% of all stroke, Cerebral hemorrhage can occur in any part of the brain parenchyma,not only can be single, but also can be multiple.Primary ICH is usually due to a long-term hypertension or cerebral amyloid angiopathy or cerebral atherosclerosis which lead the small blood vessels or small arteries to spontaneous rupture.Cerebral hemorrhage as a multifactorial disease affect by environmental factors and genetic factors epidemiological studies have shown that high blood pressure, hyperlipidemia, age, excessive alcohol consumption, smoking, Body Mass Index, diabetes are risk factors for ICH. In recent years, with the phenomenon of familial aggregation of cerebral hemorrhage was found, genetic factors in the pathogenesis of cerebral hemorrhage caused by people’s concern and become a research hotspot.In recent years, with the phenomenon of familial aggregation of cerebral hemorrhage was found, genetic factors in the pathogenesis of cerebral hemorrhage caused by people’s concern, become a research hotspot. Into the 21st century an increasing number of studies have found that NFAT family, as a based factor of many intracellular signal transduction pathway that can regulate expression levels of many factors in T cells, B cells and many of cytokines (IL-2, IL-3, IL-4, IL-5, IL-6, granulocyte macrophage colony-stimulating factor, tumor necrosis factor-a, Cyclooxygenase-2, Apob、nur77 etc.) Because TNF-a is closely related to essential hypertension and abnormal lipid metabolism and atherosclerosis, COX-2 is closely related to atherosclerosis, Apob、IL-6, nur77, SREBP-1 are closely related to abnormal lipid metabolism, NFATC1 mediates the activation of TNF-a. There are different transcription factor potential binding site of AP-1/Ets/NFAT in the TNF-a initiation factor. These transcription factors play an important role on the activation of TNF-a gene, NFATC1 mediates COX-2 expression, NFATC1 expression and COX-2 expression is positively correlated, There are SREBP-1 binding sites on exon-2 of NFATC1, So NFATC1 directly involved in SREBP-1 negative regulation.NFATC1 also mediates expression of the nur77. NFATC1 makes T cells extensively infiltrate into the intima and causes to inflammation, through mediating expression of a number of factors in T cells.Hypertension is an important independent risk factors of intracerebral hemorrhage Abnormal lipid metabolism and cerebral artery atherosclerosis are important pathological basis of cerebral hemorrhage, NFATC1 control the factors which are closely related to high blood pressure, arteriosclerosis and dyslipidemia.NFATCl is involved in intimal lesions of the inflammatory response through regulating the factoris of T cells, So we have reasons to believe that transcription factor NFATC1 is closely related to cerebral hemorrhagic stroke. At present the expression of NFATC1 in the cerebral hemorrhage and its mechanism is not yet clear, Especially there is no study about effect of polymorphisms of NFATC1 on cerebral hemorrhage at home and abroad. Thus requires us to do a lot of works to make it clear that the effect of polymorphisms of NFATC1 on cerebral hemorrhage and to explore the pathogenesis of cerebral hemorrhage from the molecular level, If this correlation exists, We can find high-risk group of cerebral hemorrhage early and execute individualized treatment through finding out the gene in the population, which can more effectively prevent and control the occurrence of cerebral hemorrhage and will bring new hope to the prevention and treatment of cerebral hemorrhage.Material and menthodA case-control design was intruduced, The subject of ICH group were consecutively recruited from northern China region patients with a total of 230 cases.male 144 and female 86, aged 33-85 years old, Han nationality, no consanguinity with each other. The controls matched by gender, age、nationality and ethnic orign were recruited from the same patients geographic region, with a total of 233 cases, male 146 and female 87. This study methods includes questionnaire survey and investigation etc. are the basic situations of the subject were recorded(gender, age, BMI, systolic blood pressure, diastolic blood pressure, addicted to tobacco and alcohol history, etc.) Hitachi 7600 automatic biochemical analyzers were used to detect CHO、TG、LDL、HDL、Apoe-a、Apoe-b of people in patients group and controls group. The functional SNPS of NFATC1 exon-2 were genotyped by Dideoxy-termination sequencing method,The variable number tandem repeat(VNTR) of intron-7 was genotyped by polymerase chain reaction analysis, The snp rs754093(Cys751Gly) of NFATC1 exon-9 was genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis. Genotyping results were confirmed by dideoxy-termination sequencing method Thex2 goodness of fit chi-square was used to test for deviation of genotype distribution from Hardy-Weinberg equilibrium, Statistical Packages for Social Sciences (SPSS 13.0) and EXCEL was used to the establish databases. The measurement data were tested for normal distribution, All statistics of normal distribution were expressed by mean±standard. Statistics of Skewed distribution were performed logarithmic conversion and then data processed.T test was used to assess difference about blood lipids and other measurement data between case group and control group,χ2 test was used to assess difference about Count data of basic background information between case group and control group Four grid table and the R×C listx2 test was used to assess difference about allele and genotype frequency between case group and control group. One-Way ANOVA was used to assess difference about serum lipid of each genotype in the case group Significant difference of statistic was considered by P< 0.05. Logistic regression analysis was used to judge risk degree of various risk factors of cerebral hemorrhage. Their respective odds ratio (OR) and 95% confidence interval (CI) were calculated. Statistical significant difference was considered by OR of 95% CI which does not contain 1.Result1.There was no statistical difference in gender, age, body mass index (BMI) between two groups(P> 0.05). It was significantly higher comparing hypertension, Diabetesmellitus, hyperlipidemia, smoking, drinking in cerebral hemorrhage group with that in the the control group,(P<0.05). There was no significant statistical difference in triglyceride (TG), apolipoprotein a (Apoa) between acerebral hemorrhage group and the control group(P> 0.05); It was significantly higher comparing the high-density lipoprotein (HDL), low-density lipoprotein (LDL), apolipoprotein b (Apob), systolic blood pressure (SBP), diastolic blood pressure (DBP) in the cerebral hemorrhage group with that in the control group, while It was significantly lower comparing CHO lesterol (CHO) in the cerebral hemorrhage group with that in the control group. There was significant difference of statistic in two groups (P<0.05).2.The genotype frequencies of all polymorphism conformed to the expectations of Hardy-Weinberg equilibrium (P> 0.05),It was suggested that the selected samples could represent the population and were suitable for genetic analysis.3.The SNPS as rs1051978, rs62096875, rsl2605457, rs1063669, rs61731547 of NFATC1 exon-2 were genotyped by Dideoxy-termination sequencing method The rs 1051978 is A/C two-states SNP, It was lower comparing A/A genotype frequencies in cerebral hemorrhage group with that in the the control group.while It was higher comparing the A/C genotype frequency in cerebral hemorrhage group with that in the the control group. Genotype frequenciesχ2=0.349, P=0.555, Allele frequenciesχ2=0.35, P= 0.661. There was no statistical difference in genotype frequencies and allele frequencies between two groups. rs62096875 is G/G homozygotes, rs 12605457 is C/C homozygous, rs 1063669 is G/G homozygotes, rs61731547 is C/C homozygotes. No new mutations were found.4.It was lower comparing variable number tandem repeat (VNTR) L/L, S/S genotype frequencies of intron-7 in cerebral hemorrhage group with that in the the control group.while It was higher comparing the L/S genotype frequency in cerebral hemorrhage group with that in the the control group. Genotype frequenciesχ2= 0.957, P= 0.620, Allele frequenciesχ2= 0.12, P= 0.727. There was no statistical difference in genotype frequencies and allele frequencies between two groups.5.It was lower comparing SNP rs754093 T/T, T/G genotype frequencies of NFATC1 exon-9 in cerebral hemorrhage group with that in the the control group, while It was significantly higher comparing the the G/G genotype frequency in cerebral hemorrhage group with that in the the control group. Genotype frequenciesχ2= 6.343, df= 2, P= 0.042; G and T/G allele frequenciesχ2= 6.39, df= 1, P= 0.011,OR= 1.411,95% CI= 1.080～1.843. There was significant difference of statistic in genotype frequencies and allele frequencies between two groups (P<0.05). 6.There was significant difference of statistic in CHO, LDL-C, Apob, among RS754093 T/G genotypes in Cerebral hemorrhage group by ANOVA. Pairwise comparison between the two groups showed that it was significantly higher comparing CHO, LDL-C, Apob in the GG genotype group with that in T/T and T/G genotypes group. There was significant difference of statistic in CHO between GG genotype and T /T and T/G genotype F=10.423, P=0.000. There was significant difference of statistic in LDL between GG genotype and T/T and T/G genotype F=23.398, P= 0.000. There was significant difference of statistic in Apob between GG genotype and T /T and T/G genotype F=32.176 P=0.000. It was higher comparing SBP in GG genotype with that in T/G genotype. It was significantly higher comparing SBP in GG genotype with that in T/T genotype. There was significant difference of statistic in SBP between GG genotype and TT genotype F=2.129, P=0.042. There was no statistical difference in diabetes, tobacco, alcohol; BMI etc among RS754093 T/G genotypes in Cerebral hemorrhage group.7.Logistic regression analysis showed NFATC1 rs754093 GG genotype is one of independent risk factors for cerebral hemorrhage (OR=3.902,95% CI 1.735～8.775, P =0.001). In addition, cerebral hemorrhage associated withThe other risk factors for cerebral hemorrhage are, LDL, Apob and systolic blood pressure.Conclution1.Hypertension, Diabetesmellitus, hyperlipidemia, Smoking, drinking are significantly associated with cerebral hemorrhage, high density lipoprotein, low-density lipoprotein, apolipoprotein b, systolic blood pressure, diastolic blood pressure, total cholesterol are significantly associated with cerebral hemorrhage.There is no significant association of Triglyceride, apolipoprotein a with cerebral hemorrhage.2.There is no significant association of the functional SNPS rs1051978, rs62096875, rs12605457, rs1063669, rs61731547 of The exon-2 with cerebral hemorrhage in Han population of Liaoning Province China.3.There is no significant association of (VNTR) L/S genotype with cerebral hemorrhage in Han population of Liaoning Province China.4.The rs754093 G/G genotype of NFATC1 exon-9 might be the independent risk factor of cerebral hemorrhage in Han population of Liaoning Province China, and its risk mainly come from G allele.5.NFATC1 gene is the susceptible gene significantly associate with cerebral hemorrhage in Han population of Liaoning Province China,in NFATC1 gene SNP rs754093 T/G allele may be a susceptible and mutational site of cerebral hemorrhage, G allele is associated with high risk of cerebral hemorrhage.6.NFATC1 rs754093 G/G genotype is significantly associated with high risk factors of cerebral hemorrhage which are CHO LDL, Apob, high systolic blood pressure.7.LDL, Apob, high systolic blood pressure are independent risk factors of cerebral hemorrhage too.更多还原