胎盘Leptin、Igf-1基因启动子区的甲基化状态与巨大儿的关联性研究

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题名	胎盘Leptin、Igf-1基因启动子区的甲基化状态与巨大儿的关联性研究
其他题名	Relationship between methylation status of placental Leptin and Igf-1 promoter and macrosomia from normal pregnancy
作者	胥新芸
学位类型	硕士
导师	闫洪涛
答辩日期	2015-05-24
学位授予单位	温州医科大学
学位专业	遗传学
关键词	巨大儿 LEP 胎盘 Igf-1 甲基化妊娠
摘要	目的：探讨正常妊娠状态下，巨大儿胎盘瘦素（Leptin，LEP）、胰岛素样生长因-1（Igf-1）基因启动子的甲基化状态和巨大儿之间的关系，为研究巨大胎儿的发病机制及其临床防治措施提供依据。资料与方法:研究对象研究人群来自2011年4月~2012年3月在温州医学院附属第二医院产科分娩的101例新生儿及母亲。入选标准：正常妊娠、单胎足月分娩、糖耐量试验正常、所产新生儿无先天畸形的产妇。胎儿娩出后立即称重，并对其发育状况进行评价，分为巨大儿组（新生儿体重≥4000g，n=48）和正常体重儿组（2500g≤新生儿出生体重＜4000g，n=53）。方法（1）胎盘标本的采集和处理：胎盘娩出后，无菌采集胎盘滋养层组织1cm3，剪碎放入5倍体积组织的RNAlater中， 4孵化过夜，随后转入-80保存，待DNA和RNA的提取。（2）临床资料的收集：采用自行设计的调查表，收集产妇和新生儿相关资料。主要内容包括：1）新生儿发育情况；2）产前检查情况；3）孕期营养状况；4）分娩情况；5）新生儿父母亲的身高、体重、吸烟、饮酒等资料。（3）甲基化和表达的的测定：采用Sequenom MassARRAY Methylation平台检测LEP和Igf-1 基因启动子的甲基化状态；实时荧光定量PCR（Real-time PCR）检测基因LEP和Igf-1 的mRNA表达。（4）统计分析：问卷调查资料和实验室数据统一录入EpiData数据库。采用SPSS 17.0统计软件进行统计描述与统计推断。统计方法主要采用方差分析、独立样本t检验、秩和检验等。结果（1）人口统计学资料显示：两组中母亲年龄、妊娠周期，婴儿的性别等主要人口学特征均未见统计学差别；巨大儿组母亲的孕前体重略高（p=0.049）；巨大儿的分娩更倾向于采用剖宫产手术（p=0.003）。（2）胎盘LEP的甲基化和mRNA水平在两组中的比较：巨大儿组的LEP基因启动子区的平均甲基化水平（35.6%）高于正常体重儿儿组（34.6%），但没有统计学意义（p = 0.538)，且单个CpG二核苷酸的甲基化程度和mRNA的表达在两组中也无差异。当按照胎次分层时，初产妇中巨大儿LEP启动子区的个别CG片段的甲基化水平高于正常组，并且有统计学意义。当孕周为39周时，LEP启动子区的个别CG位点和启动子的平均甲基化水平在两组中有统计学差异，且巨大儿组高于正常体重儿组，但这些甲基化的变异没有影响mRNA的表达。（3）胎盘Igf-1的甲基化和mRNA水平在两组中的比较：巨大儿组Igf-1 P1启动子区的平均甲基化水平高于正常体重儿组（36.1% vs. 33.9%），但没有统计学意义（p = 0.453), 且单个的CG位点的甲基化水平比较没有统计学差异。Igf-1 mRNA的表达在总体和分层分析中都未发现统计学差异。结论：在巨大儿发生过程中，胎盘LEP的甲基化可以受到母体环境或特定孕龄的影响，而胎盘Igf-1 P1启动子区的甲基化状态和巨大儿的发生无关。
其他摘要	OBJECTIVE To investigate whether the DNA methylation status and mRNA expression level of the LEP gene and Igf-1 gene is altered in the human placenta of macrosomic infants with normal pregnancy, and providing evidence for the study of the pathogenesis of fetal macrosomia and its clinical PreventionMaterial and Methods:Source A total of 49 neonates, who were born with high birth weight (≥ 4000 g), or a condition called macrosomia, on normal pregnancy, and 52 neonates with normal birth weight (control group) (2500 g < birth weight < 4000 g) were recruited from the Second Affiliated Hospital of Wenzhou Medical College, China. Case seleetion criteria: maternal health, were singleton on pregnancy, had no history of hypertension, gestational diabetes (including normal glucose tolerance tests during the first and third trimesters of pregnancy). No fetal distress, congenital malformations. Methods （1）Placental sampling and handlingPlacenta biopsies were obtained from the mothers considered at term after delivery. The biopsies, approximately 1 cm3, were obtained evenly from the center of the cotyledons across the placenta, were cut into small pieces, transferred to 5 volumes of the RNAlater solution (Ambion, Austin, Texas), and incubated at 4 °C overnight, freezing at –80 °C until nucleic acid extraction.（2）Clinical data collecting Collecting the medical information of neonates and mothers using the self designed questionnaire, including: 1）the development of neonates ; 2）the condition of prenatal examination; 3）the nutritional condition during pregnancy; 4）delivery situation; 5）the height, weight, smoking and drinking status of father and mother 1.2.3 Measurement of DNA methylation and mRNA expressionThe DNA methylation status of LEP and Igf-1 promoter and the gene expression levels of the placenta were determined using the Sequenom Mass ARRAY and real-time PCR, respectively.（3）Statistical analysisThe data from the questionnaire were imported to the EpiData。The data was statistics bysofeware SPSS 17.0, including using One-way ANOVA, unpaired t-test, Mann-Whitney rank sum test.Results（1）Sample characteristics：The distributions of gestational age, infant gender and maternal age used were not significantly different between the groups. As expected, Women with higher body weights prior to pregnancy tend to gave birth to macrosomic babies (p =0.049), through cesarean section (p=0.003). （2）Comparation of methylation and mRNA level of placental LEP between the two groups：No significant difference of mean or individual CpG site DNA methylation in LEP gene promoter was observed between the macrosomia and normal weight babies (mean level：35.6% vs.34.6 %, p=0.535 ), as well as the LEP mRNA expression level. in nullparity women, we have shown that LEP gene DNA methylation status is altered at several CpG sites in promoter between macrosomia and normal weight babies( p<0.05）. And also the mean and individual CpG sites methylation level of macrosomia is significant higher than control group when the gestational age is 39 wks（mean level: n=14/17, 38% vs.32%, p=0.016）, both differences do not contribute to the expression level of LEP in placenta. （3）Comparation of methylation and mRNA level of placental Igf-1 between the two groups: compared to the normal birth weight group, the methylation mean level of Igf-1 P1 prompter have a higher trend in the macrosomia, but without statistical significance (36.1% vs. 33.9%) nor the individual CG sites. The Igf-1 mRNA level shows no difference between the two groups even after stratified analysis. Conclusion: The methylation status of the placental LEP promoter in macrosomia from normal pregnancy can be altered within a specific gestational period or by certain maternal conditions, but placental LEP expression is not affected. The placental Igf-1 has no contribution to macrosomia from normal pregnancy
语种	中文
学号	2010010005
发布年限	2010-05-27
毕业论文分类号	0R03
原始专题	环境与公共卫生学院
学位论文研究方向	表观遗传学
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全文文件名	2010010005胥新芸2013遗传学.pdf\|2010010005胥新芸2013遗传学.pdf
文献类型	学位论文
条目标识符	https://kms.wmu.edu.cn/handle/3ETUA0LF/117161
专题	温州医科大学
作者单位	公共卫生与管理学院
推荐引用方式 GB/T 7714	胥新芸. 胎盘Leptin、Igf-1基因启动子区的甲基化状态与巨大儿的关联性研究[D]. 温州医科大学,2015.