职业性铝接触与组蛋白赖氨酸甲基化修饰关系的研究

发布时间：2018-04-15 21:39

本文选题：铝 + 认知功能　；参考：《山西医科大学》2017年硕士论文

【摘要】：目的:本次研究采用分子流行病学方法在职业性铝接触人群中开展研究,评估长期职业性铝接触人群认知功能改变情况,分析血铝与外周血淋巴细胞H3K4me3、H3K9me2、H3K27me3及血浆BDNF、EGR1蛋白水平之间的关系,初步探讨长期职业性铝接触引起的组蛋白赖氨酸甲基化谱改变情况,为长期铝暴露的分子流行病学研究提供参考依据,并为以后研究上述指标是否可作为从外周血水平对铝的神经毒性进行监测和早期诊断的替代生物标志物提供人群分子流行病学依据。方法:采用整群抽样的方式,选择山西某铝厂电解铝车间100例男性工人作为暴露组、热电车间135例男性工人作为对照组。采用自行设计的职业流行病学调查问卷:简短精神状态量表(MMSE)、数字广度测试[DST,包括正序测试(DSFT)和倒序测试(DSBT)]、画钟试验(CDT)、物体记忆测验(FOME)、言语流畅性测验(VFT)等进行调查,收集研究对象的一般情况并进行认知功能评分;采集调查对象的空腹肘静脉血液,分离血浆并提取淋巴细胞,-80℃冻存备用;采用石墨炉原子吸收法测定血铝含量,可能由于该大型铝厂对当地环境的影响,对照组和暴露组工人的血铝水平无统计学差异,故以血铝水平的上下百分位数P25、P75和中位数M将研究对象分为低、中、高血铝组;酶联免疫吸附试验(ELISA)方法定量检测淋巴细胞中H3K4me3、H3K9me2、H3K27me3表达水平和血浆BDNF、EGR1蛋白水平。结果:1.职业性铝接触工人认知功能与血浆BDNF、EGR1的关系:(1)职业性铝接触工人的血铝水平比较:接触组工人血铝值为(155.07±71.17)μg/L,对照组工人血铝值为(142.94±53.80)μg/L,两组差异无统计学意义(F=1.488,P0.05)。下四分位数P25为100.14mg/L、中位数M为134.36mg/L、上四分位数P75为178.96mg/L,故按照血铝水平的四分位数:m(p25,p75)为134.36(100.14,178.96)mg/l,将所有研究对象分为低血铝组82.88(72.69,92.40)mg/l、中血铝组134.46(115.74,154.68)mg/l和高血铝组228.59(204.91,263.87)mg/l。(2)不同血铝水平工人的基本情况比较:不同血铝水平工人在年龄、工龄、受教育程度及吸烟、饮酒方面,差异均无统计学意义(均p0.05)。(3)不同血铝水平工人的认知功能评价:不同血铝水平工人的mmse、dsft、dsbt、dst得分不同,差异均有统计学意义(均p0.05)。高血铝组工人mmse、dsft、dsbt、dst的平均得分均分别低于低血铝组工人的平均得分,差异均有统计学意义(均p0.05);中血铝组工人mmse、dsbt、dst的平均得分均分别低于低血铝组工人的平均得分,差异均有统计学意义(均p0.05)。(4)血铝与认知功能的相关分析结果显示:血铝水平和认知功能之间具有不同程度的相关性。血铝水平与mmse评分之间存在负相关(rs=-0.252,p0.01),与dsft评分之间存在负相关(rs=-0.197,p0.01),与dst评分之间存在负相关性(rs=-0.181,p0.01),说明随着血铝水平的升高,认知功能评分中的mmse、dsft、dst得分均呈降低趋势。(5)影响认知功能的多因素回归分析结果显示;血铝、年龄、工龄、受教育程度分别被纳入回归方程,差异有统计学意义。说明年龄、工龄、受教育程度均可能影响认知功能。(6)工人血浆bdnf、egr1蛋白水平分层分析结果显示:不同血铝组工人血浆bdnf蛋白(f=5.385,p=0.005)表达量、egr1蛋白(f=6.853,p=0.001)表达量不同,差异有统计学意义。与低血铝组相比,高、中血铝组工人血浆bdnf表达量均分别降低,差异均有统计学意义(均p0.05)。高血铝组工人血浆egr1蛋白表达量分别低于低、中血铝组工人血浆egr1蛋白表达量,差异均有统计学意义(均p0.05)。但是按照年龄、工龄、受教育程度分层分析后,结果均显示:血浆bdnf、egr1蛋白水平在各组间表达量差异并无统计学意义。(7)血铝、bdnf、egr1蛋白及认知功能的相关性分析结果:血铝水平、bdnf、egr1和认知功能之间具有不同程度的相关性。血铝水平与bdnf蛋白、egr1蛋白水平之间均存在负相关性(r=-0.214,p0.01、r=-0.315,p0.01),说明随着血铝水平的升高,bdnf和egr1的表达量均呈下降趋势;bdnf蛋白水平与mmse评分之间存在正相关性(rs=0.140,p0.05),egr1蛋白水平与mmse评分之间存在正相关性(rs=0.201,p0.05),说明随着bdnf、egr1的表达量降低,mmse评分呈降低趋势。2.职业性铝接触工人组蛋白赖氨酸甲基化修饰改变及影响分析:(1)不同血铝水平工人外周血淋巴细胞h3k4me3水平比较:不同血铝水平工人外周血淋巴细胞h3k4me3的表达量不同,差异有统计学意义(f=3.572,p=0.031)。高血铝组工人h3k4me3表达量低于低血铝组h3k4me3表达量,差异有统计学意义(p0.05);高血铝组h3k4me3表达量低于中血铝组h3k4me3表达量,中血铝组h3k4me3表达量低于低血铝组h3k4me3表达量,但差异均无统计学意义(均p0.05)。(2)不同血铝水平工人外周血淋巴细胞h3k9me2水平比较:不同血铝水平工人外周血淋巴细胞h3k9me2的表达量不同,差异有统计学意义(f=9.551,p=0.001)。高血铝组工人h3k9me2表达量高于低血铝组和中血铝组工人h3k9me2的表达量,差异均有统计学意义(均p0.05);中血铝组工人h3k9me2表达量高于低血铝组工人h3k4me3表达量,差异有统计学意义(p0.05)。(3)不同血铝水平工人外周血淋巴细胞h3k27me3水平比较:不同血铝水平工人外周血淋巴细胞h3k27me3的表达量不同,差异有统计学意义(f=3.641,p=0.028)。高血铝组工人h3k27me3的表达量高于低血铝组工人h3k27me3的表达量,差异有统计学意义(p0.05)。(4)淋巴细胞h3k4me3、h3k9me2、h3k27me3与血铝的相关性分析:h3k4me3、h3k9me2、h3k27me3表达量和血铝之间存在不同程度的相关性。其中h3k4me3和血铝之间呈负相关(r=-0.307,p0.01),说明随着血铝水平的升高,职业性铝接触工人外周血淋巴细胞h3k4me3表达量降低;h3k9me2、h3k27me3与血铝之间均呈正相关(r=0.329、r=0.387,均p0.01),说明随着血铝水平升高,职业性铝接触工人外周血淋巴细胞h3k9me2、h3k27me3表达量也升高。(5)bdnf、egr1和h3k4me3、h3k9me2、h3k27me3的相关分析结果:bdnf、egr1蛋白水平和h3k4me3、h3k9me2、h3k27me3表达量之间存在不同程度的相关性。其中bdnf、egr1和h3k4me3之间均呈正相关(r=0.202,p0.05、r=0.361,p0.01),说明随着h3k4me3表达量的降低,bdnf、egr1两种蛋白的表达量也降低;bdnf和h3k9me2之间呈负相关(r=-0.286,p0.01),说明随着h3k9me2表达量升高,bdnf的表达量降低;egr1和h3k27me3之间呈负相关(r=-0.241,p0.05),说明随着h3k27me3表达量升高,egr1蛋白的表达量降低。(6)血浆bdnf、egr1蛋白影响因子的多元逐步回归分析:bdnf与h3k4me3、h3k9me2之间存在线性回归关系,h3k9me2对bdnf的影响比h3k4me3对bdnf的影响大;egr1与h3k4me3、h3k27me3之间存在线性回归关系,h3k4me3对egr1的影响比h3k27me3对egr1的影响大。结论:1.长期职业性铝接触可引起工人认知功能改变和血浆BDNF、EGR1水平降低,且血铝、认知功能和BDNF、EGR1之间存在一定的相关性,提示铝可能通过影响BDNF、EGR1的表达影响认知功能。2.血铝水平、BDNF、EGR1和H3K4me3、H3K9me2、H3K27me3的表达水平之间均有一定的相关性,表明在职业性铝接触引起的认知功能改变过程中,铝可能通过下调H3K4me3、上调H3K27me3及H3K9me2、下调记忆相关蛋白BDNF和EGR1的表达引起认知功能改变。
[Abstract]:Objective: This study adopts the methods of molecular epidemiology research in occupation of aluminum exposed, the change of occupation of aluminum exposed population to assess the long-term cognitive function, analysis of blood aluminum and peripheral blood lymphocyte H3K4me3, H3K9me2, H3K27me3 and BDNF in plasma, the relationship between EGR1 protein level and preliminary study of aluminum exposure caused by long-term occupation histone lysine methylation profiles, provide a reference basis for the molecular epidemiology of long-term exposure to aluminum, and to investigate whether these indexes can provide the basis for molecular epidemiology as surrogate biomarkers from the peripheral blood level of neurotoxicity of aluminum for monitoring and early diagnosis. Methods: cluster the way of sampling, choose a Shanxi aluminum electrolytic aluminum workshop of 100 male workers as exposure group, 135 cases of male power workshop workers as control group. The self set. Occupation questionnaire meter: Mini Mental State Scale (MMSE), digital span test [DST, including positive test (DSFT) and reverse (DSBT) test, clock drawing test (CDT), object memory test (FOME), verbal fluency test (VFT) for investigation, cognitive function the score generally collected and venous blood collection; investigation, separation and extraction of plasma cells, -80 C cryopreserved blood; Determination of aluminium content by graphite furnace atomic absorption spectrometry may be due to the effect of the large aluminum in the environment, no significant difference between the control group and the group of workers exposed to blood aluminum level, so the blood aluminum level on the percentile of P25, P75 and M of the median object is divided into low, high blood aluminum group; enzyme linked immunosorbent assay (ELISA) method for quantitative detection of H3K4me3 in H3K9me2 cells, the expression level of H3K27me3 and blood Plasma BDNF, EGR1 protein level. Results: 1. of workers exposed to aluminum occupation cognitive function and plasma BDNF, EGR1: (1) comparison of blood aluminum level occupation of aluminum exposed workers: exposed workers blood aluminum value (155.07 + 71.17) g/L, group of workers blood aluminum value as control (142.94. 53.80) g/L, with no significant difference between two groups (F=1.488, P0.05). Four digit P25 100.14mg/L, median M 134.36mg/L, four digit P75 178.96mg/L, so in accordance with the four percentile of blood aluminum level: m (P25, p75) 134.36 (100.14178.96) mg/l, all of the subjects were divided into low blood aluminum group 82.88 (72.69,92.40) mg/l in blood aluminum group 134.46 (115.74154.68) mg/l and high blood aluminum group 228.59 (204.91263.87) mg/l. (2) compared to the basic situation of workers of different blood aluminum level: Blood aluminum level in workers of different age, seniority, education, and smoking, drinking. There were no significant differences ( P0.05). (3) to evaluate the cognitive function of workers of different blood aluminum level: workers in different blood aluminum level of MMSE, DSFT, dsbt, DST scores, the differences were statistically significant (P0.05). High blood aluminum group DSFT, dsbt, MMSE workers, the average score of DST were significantly lower than those of the average score of low blood aluminum group of workers, the differences were statistically significant (P0.05); blood group dsbt, MMSE aluminum workers, the average score of DST were significantly lower than those of the average score of low blood aluminum group of workers, the differences were statistically significant (P0.05). (4) the correlation analysis and cognitive function of aluminum. The result showed that the correlation with blood between different levels of functional and cognitive level of aluminum blood. There was a negative correlation between blood aluminum level and MMSE score (rs=-0.252, P0.01), and there was a negative correlation between DSFT scores (rs=-0.197, P0.01), and there is a negative correlation between DST scores (rs=-0.181, P0.01), with elevated blood levels of aluminum,. Know the function score in MMSE, DSFT, DST score decreased. (5) regression analysis showed that the influencing factors of cognitive function; blood aluminum, age, seniority, education level were included in the regression equation, the difference was statistically significant. The age, seniority, education level can affect cognitive function (6) workers. Plasma levels of BDNF, EGR1 protein level stratified analysis showed that different groups of workers blood aluminum plasma BDNF protein (f=5.385, p=0.005) expression of EGR1 protein (f=6.853, p=0.001) expression is different, the difference was statistically significant. Compared with low blood aluminum, high blood aluminum plasma BDNF group of workers the expression amount was decreased, the differences were statistically significant (P0.05). High blood aluminum group workers plasma EGR1 protein expression were lower in blood plasma, aluminum group of workers the expression of EGR1 protein, the differences were statistically significant (all P0.05). But according to the age, length of service, by Analysis of education level stratification, results showed that plasma BDNF expression was no significant difference in EGR1 protein levels in each group. (7) blood aluminum, BDNF, correlation between EGR1 protein and cognitive function analysis results: Blood aluminum level, BDNF, has a different degree of correlation between EGR1 and blood aluminum level of cognitive function. With the BDNF protein, there was a negative correlation between EGR1 protein level (r=-0.214, P0.01, r=-0.315, P0.01), with elevated blood aluminum level, expression of BDNF and EGR1 showed a downward trend; there is a positive correlation between BDNF protein level and MMSE score (rs=0.140, P0.05), there is a positive correlation between EGR1 protein level with the score of MMSE (rs=0.201, P0.05), with BDNF, the expression of EGR1 was decreased and the MMSE score decreased.2. occupation aluminum workers exposed to histone lysine methylation modification and analysis: (1) effects of different blood levels of aluminum industry Comparison of h3k4me3 in human peripheral blood lymphocytes of peripheral blood lymphocyte h3k4me3 expression level: workers with different amount of blood aluminum level is different, the difference was statistically significant (f=3.572, p=0.031). The high expression of h3k4me3 blood group of aluminum workers is lower than the low expression of h3k4me3 in blood aluminum group, the difference was statistically significant (P0.05); high blood aluminum group the expression of h3k4me3 was lower than that in blood group h3k4me3 expression in aluminum, aluminum blood group h3k4me3 expressed lower than low blood aluminum group the expression of h3k4me3, but the differences were not statistically significant (P0.05). (2) comparison of workers of different blood aluminum level H3K9me2 levels in peripheral blood lymphocytes of peripheral blood lymphocytes: the expression of H3K9me2 in different blood aluminum workers the amount of different levels, the difference was statistically significant (f=9.551, p=0.001). The high blood aluminum workers H3K9me2 expression group was higher than that of low blood aluminum group and blood aluminum workers H3K9me2 group, the differences were statistically significant (P0.05); blood Aluminum Group The workers H3K9me2 was higher than that of low blood aluminum group of workers h3k4me3 expression, the difference was statistically significant (P0.05). (3) comparison of workers of different blood aluminum level H3K27me3 levels in peripheral blood lymphocytes of peripheral blood lymphocyte H3K27me3 expression: workers with different amount of blood aluminum level is different, the difference was statistically significant (f=3.641, p=0.028). Expression of high blood aluminum group of workers is higher than the H3K27me3 H3K27me3 aluminum low blood group of workers, the difference was statistically significant (P0.05). (4) h3k4me3 lymphocytes, H3K9me2, correlation analysis of H3K27me3 and blood aluminum: h3k4me3, H3K9me2, there are different degrees of correlation between blood volume and the expression of H3K27me3 was negatively related to aluminum. Between the h3k4me3 and blood aluminum (r=-0.307, P0.01), with elevated blood aluminum level, occupation of aluminum exposed workers peripheral blood lymphocyte h3k4me3 expression decreased; H3K9me2, a positive correlation between H3K27me3 and blood aluminum (r=0. 329, r=0.387, P0.01), with blood aluminum level increased, H3K9me2 in peripheral blood lymphocytes of workers exposed to aluminum occupation, the expression of H3K27me3 was also increased. (5) BDNF, EGR1 and h3k4me3, H3K9me2, correlation analysis results of H3K27me3: BDNF, EGR1 protein level and h3k4me3, H3K9me2, there are different degrees of correlation between the expression of H3K27me3. BDNF, EGR1 and h3k4me3 were positively correlated (r=0.202, P0.05, r=0.361, P0.01, h3k4me3) with reduced expression of BDNF, EGR1, the expression of two proteins decreased; there was a negative correlation between BDNF and H3K9me2 (r=-0.286, P0.01), with the expression of H3K9me2 increased, the expression of BDNF was decreased; there was a negative correlation between EGR1 and H3K27me3 (r=-0.241, P0.05), with H3K27me3 expression increased, EGR1 protein expression decreased. (6) plasma BDNF, stepwise regression analysis of EGR1 protein factors: BDNF and h3k4me3, h3k yuan There is a linear regression relationship between 9me2, the effect of H3K9me2 on the BDNF than the effect of h3k4me3 on BDNF; EGR1 and h3k4me3, there was a linear regression relationship between H3K27me3 and the effect of h3k4me3 on the EGR1 than the effect of H3K27me3 on EGR1. Conclusion: 1. long-term occupation of aluminum exposure may induce changes in cognitive function and plasma BDNF, EGR1 level reduced, and blood aluminum, cognitive function and BDNF, there is a certain correlation between EGR1, suggesting that aluminum may influence the BDNF, the expression of EGR1 on cognitive function in.2. blood aluminum level, BDNF, EGR1 and H3K4me3, H3K9me2, have a certain degree of correlation between the expression level of H3K27me3, showed that cognitive function caused by occupation of aluminum contact the change in the process of aluminum through down-regulation of H3K4me3 and up regulation of H3K27me3 and H3K9me2, downregulation of memory related proteins BDNF and EGR1 caused changes in cognitive function.

【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R135

【参考文献】