【作者】 温世宝；

【导师】 巴月；

【作者基本信息】 郑州大学 ， 劳动卫生与环境卫生学， 2012， 硕士

【摘要】 DNA甲基化是肿瘤的主要危险因素之一,且许多研究结果显示DNA甲基化模式在胎儿出生时已经形成,印记基因H19(imprinted maternally expressed transcript (non-protein coding))与多种肿瘤的发生相关,在胚胎期表达活跃,且主要受基因甲基化的控制,所以探索生命早期影响胎儿表观遗传(DNA甲基化)的因素,阐明H19甲基化与孕期环境及营养暴露之间的关系,可为肿瘤的预防及早期营养干预提供新的思路。目的：探索孕期血液一碳单位营养物(叶酸、维生素B12)、锌以及环境暴露与H19基因甲基化模式及新生儿出生特征之间的关系,为在胎儿期通过营养手段预防肿瘤提供线索。材料与方法：1.调查对象的选择：以2008年4月到2008年9月期间在郑州市侯寨中心卫生院分娩的、未患乙肝、糖尿病及其它慢性消化道疾病的(以临床诊断为准)健康母亲作为调查对象,提前告知本课题目的意义并签署知情同意书后,共99名母亲纳入调查范围。2.资料的收集：根据此次研究的内容详细设计调查问卷(见附件),严格培训调查员并实施预调查后正式开始问卷调查,调查对象的个人信息均严格保密；收集2007年5月到2008年9月当地环保局公布的每周空气污染指数及分指数。3.新生儿出生特征的测量：用头围尺经颅骨两眼眉弓及枕骨结节测量新生儿头围,用卧式身长计测量新生儿出生身长；用电子秤称量新生儿出生体重。4.血清中一碳单位营养物及锌的测定：采用Access磁微粒化学发光免疫分析系统测定血清中叶酸、维生素B12；原子吸收法测定血清锌水平。5.甲基化水平的测定：甲基化特异性荧光定量PCR (quantitativemethylation-specific polymerase chain reaction, QMS-PC R)测定H19基因启动子及差异甲基化区(different methylation region, DMR)甲基化率。6.数据整理和分析：数据录入采用Epidata3.1软件并采用SPSS12.0进行分析。主要分析检验方法有：配对t检验、多元线性回归分析等,检验水准α=0.05。结果：1.新生儿出生特征、母体血清营养物含量及产妇孕期环境因素的关系：(1)新生儿共99例,男孩52例,女孩47例,出生身长为49.48±1.89cm,出生体重为49.48±1.89kg,出生头围为33.83±1.19cm；多元回归分析表明：新生儿出生体重与新生儿性别相关(β=0.198,P=0.046)；出生身长与产妇剖宫产相关(β=0.283,P=0.008)。出生体重、出生身长及出生头围分别与产妇孕前体质指数(body mass Index,BMI)相关(β=-0.198,P=0.046；β=0.336,P=0.003；β=0.314, P=0.012):(2)产妇静脉血血清及新生儿脐带血血清叶酸水平分别为2.29±2.13ng/ml和7.29±4.80ng/ml,锌水平分别为0.67±0.31μg/ml和0.93±0.37μg/ml,维生素B12水平分别为175.34±154.78pg/ml和240.19±228.56pg/ml,其中,新生儿脐带血血清锌水平高于产妇(t=-5.551,P=0.001),但未发现二者具有相关关系：新生儿脐带血血清叶酸水平明显高于产妇血清叶酸水平(t=-12.183,P=0.001),且二者相关(r=0..367,P=0.001)；新生儿脐带血血清维生素B12水平高于产妇(t=-4.432,P=0.001),且二者具有相关性(r=0.325,P=0.001)。服用保健品组与未服用保健品组三种营养素水平差异均无统计学意义(P＜0.05)。2.产妇静脉血血清和新生儿脐带血血清—碳单位营养物水平、母亲孕期环境因素与H19甲基化模式的关系：(1)母血H19启动子甲基化率均值为68.53%,脐血为68.00%；两者差异无统计学意义(t=-0.256,P=0.798),且两者之间呈正相关关系(β=0.322,P=0.002)；母血H19DMR甲基化率均值为83.01%,脐血为83.25%；两者差异无统计学意义(t=-0.105,P=0.917),且两者之间呈正相关关系(β=0.472,P=0.001)。(2)新生儿血清H19DNA启动子甲基化水平与孕期平均每周PM10污染分指数负相关(β=-1.559,P=0.049)；新生儿H19的DNA启动子甲基化状态与新生儿血清锌具有相关性(β=0.275,P=0.009)；脐带血H19DMR甲基化水平分别与脐血中叶酸水平,母亲被动吸烟和剖宫产相关(β=0.310,p=0.036；β=0.334, P=0.005；β=-0.284,P=0.018)；产妇H19DMR甲基化状态与产妇血清锌相关(β=0.420,P=0.008)。(3)未发现新生儿H19启动子的甲基化状态与产妇血清叶酸、维生素B12、孕期体重增加、被动吸烟、孕妇年龄等因素相关；也未发现产妇H19启动子的甲基化状态与血清叶酸,维生素B12、被动吸烟、孕期体重增加、空气质量等因素相关。结论：1.产妇孕前BMI可影响新生儿的出生体重、出生身长和出生头围。2.新生儿脐血中H19启动子、DMR甲基化率主要取决于母亲血液中H19基因的甲基化率。3.产妇静脉血及新生儿脐血H19甲基化率受血液中营养物质及环境因素的共同影响。更多还原

【Abstract】 DNA methylation is a kind of important factor in the progress of cancer. Many researchers have demonstrated that gene methylation model is established in fetus; Imprinted gene H19is transcripted highly in fetus, mainly controlled by gene methylation. It has been verified that it is related with many tumors. Thus, to explore the factors affecting the express of epigenetic gene, such as DNA methylation, and clarify the role of environment and nutrition in the establishment of methylation will provide an important clue for the prevention of cancer.Objective:The aim of this study was to explore the relationship among "maternal nutritional intake, environmental factors and H19DNA methylation patterns in offsprings and birth characteristics", and to provide basic data for cancer prevention and nutritional intervention.Materials and methods:1. Selection of subjectsPregnant women who came to houzhai hospital for delivery between April and September2008were recruited into this study. Any one with diabetes, hepatitis B infection or other coeliac diseases was not included. Total99pregnant women were recruited as subjects and willing to participate in the study after being informed the prupose of the study.2. Questionnaire was designed according to the purpose of this study. All investigator were trained strictly and conducted pre-investigation to make sure the success of the investigation. All the information was kept secret. Air pollution data of local region was collected through Environmental Protection Bureau.3. Determination of birth characteristicsThe birth weight of infants was weighed using electronic scale; The head circumference and the birth length of offsprings were measured using horizontal length meter. 4. Determination of one-carbon nutrients in serumSerum folate and vitamin B12was determined by paramagnetic particle chemiluminescent immunoassay method. Serum zinc was determined by Atomic absorption spectrophotometry.5. The determination of H19DNA methylation status Methylation patterns of H19promotor and H19DMR (Different methylation region) were tested by QMS-PCR (quantitative methylation-specific polymerase chain reaction)6. Data management and statistical analysisAll the data was entering using software of Epidata3.1and furtherly analysed by the SPSS software, version12.0. Following methods were used in the study:paired sample T-test, multiple linear regression analysis and so on. A P-value of less than0.05was considered statistically significant.Results:1. The relationship among birth characteristics, nutrient content of maternal serum and maternal environmental exposures during pregnancy.(1)The average birth lengh of infants was49.48cm, the average birth weight of infants was3.19kg, the average birth head circumference of infants was33.83cm; multiliner regressive modle showed that birth lengh, birth weight and birth head circumference were related with BMI (body mass index) of mother (β=-0.198, P=0.046;β=0.336,P=0.003;β=0.34, P=0.012); Birth weight was related with infant genders (β=-0.198, P=0.046); Birth lengh was related with maternal cesarean (β=0.283, P=0.008)。(2)The average serum folate of mother and infants was29ng/ml and7.29ng/ml respectively; The average serum vitamin B12of mother and infants was175.34pg/ml and240.19pg/ml respectively; The average serum zinc of mother and infants was0.67μg/ml and0.93μg/ml respectively。 Serum folate level, vitamin B12and Zinc in cord blood was significantly higher than that in mother’s blood respectively (t=-12.183, P=0.001;t=-4.432, P=0.001; t=-5.551, P=0.001); Serum folate level and vitamin B12in cord blood was correlated with that of mother’s blood repectively (r=0.367,P=0.001; r=0.325, P=0.001); Serum Zinc level in cord blood was not correlated with that of mother’s blood(P>0.05).2.The relationship of serum nutrient content,maternal environmental exposures alld H19DNA methylation pattern.(1)The average percentages of methylation in H19promoter were68.53%for mother’s blood and68.00%for cord blood respectively.There was no significant difference of methylation in H19promoter between cord b1ood and mother’s b1ood (t=0.256,P=0.798),but correlated with each other(β=0.275,P=0.009).There was no significant difference of methylation in H19DMR between cord blood and mother’s blood(t=-0.105,P=0.917),but correlated with each other(β=0.472,p=0.001)(2)H19DNA promoter methylation status ofmaternal was correlated with cord blood serum zinc (β=0.275,P=0.009)；H19DMR methylation status of infants was correlated with vitamin B12of infants,maternal cesarean and passive smoking respectively(β=0.310,P=0.036；β=-0.334,P=0.005；β=-0.284,P=0.018)；H19DMR methylation status of maternal was correlated with maternal serum zinc (β=-0.420,P=0.008)(3)No correlation was observed between methylation in H19promoter of cord blood and maternal serum folate,vitalnin B12,weight gain during pregnanc,mother’s age,air pollution；Neither was methylation in H19promoter of maternal blood.Conclusion:1.Maternal pre-pregnancy body mass index is related with birth characteristics.2.Methylation of cord blood mainly depelldents on maternal Serum Hl9methylation.3.H19methylation is affected by nutrient of blood and environmental exposures更多还原