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学科主题: 妇产科学
题名:
子痫前期滋养细胞线粒体LCHAD基因甲基化与基因表达相关性的
作者: 孟然
答辩日期: 2016-11-21
导师: 杨孜
专业: 妇产科学
授予单位: 北京大学
授予地点: 北京大学第三临床医学院
学位: 硕士
关键词: 长链脂肪酸氧化 ; 子痫前期 ; 滋养细胞 ; DNA甲基化
其他题名: Study of the relevance of DNA methylation with the gene expression of mitochondrial LCHAD in trophoblast cells of preeclampsia
分类号: R714.24
摘要:

目的

1. 探讨长链游离脂肪酸对胎盘滋养细胞线粒体β氧化循环长链3-羟基酰基辅酶A脱氢酶(LCHAD)基因启动子区甲基化程度影响,以及对甲基化修饰的时间效应。

2. 探讨早发重度子痫前期、HELLP综合征及抗磷脂综合征(APS)对胎盘滋养细

胞线粒体LCHAD基因甲基化程度的影响及其与基因mRNA表达的相关性。

方法

第一部分:

1. 体外培养原代人绒毛滋养细胞及HTR-8/SVneo永久性人滋养细胞,研究组采用长链游离脂肪酸孵育滋养细胞(LC-FFA组),并以短链游离脂肪酸(SC-FFA组)和中链游离脂肪酸(MC-FFA组)做为不同链长游离脂肪酸对照组,空白对照组无脂肪酸(F-FFA组)。分别在孵育24、48及72h收集细胞并提取DNA。

2. 在LCHAD基因转录起始位点上游2000bp区域内序列预测CpG岛位置,设计甲基化检测位点和引物。

3. 采用Sequenom MassArray甲基化DNA定量分析平台检测不同组CpG岛各CpG位点的甲基化程度后进行各甲基化位点和CpG岛的统计学分析。

 

 

第二部分:

采集2013年1月至2015年3月在北京大学第三医院产科检查及住院治疗的

   孕28-32周病理妊娠孕妇的血清,其中早发重度子痫前期 14 例(子痫前期

   组)、HELLP 综合征 12 例 (HELLP组)及APS 14例(APS组)为病理妊娠

   组,以正常妊娠的14例孕妇的血清为对照(对照组);原代绒毛滋养细胞来

   自于2014年3月至2014年8月在北京大学第三医院产科门诊自愿要求人工

   流产、胚胎正常、无妊娠合并症和并发症妇女40例的绒毛组织。在体外培养

   原代绒毛滋养细胞和永久性滋养细胞系HTR-8/Svneo细胞,分别加入相同浓

   度(10%)的4组孕妇的血清,孵育24 h后收集各组滋养细胞并提取DNA。

2. 通过MassARRAY平台检测4组滋养细胞中LCHAD 基因启动子区各位点的甲基化程度。

3. 采用实时荧光定量逆转录(RT)-PCR技术检测4组滋养细胞中LCHAD mRNA的表达水平。

4. 采用Pearson相关系数对4组滋养细胞中LCHAD基因启动子区各位点的甲基化程度与 LCHAD mRNA 的表达水平进行相关性分析。

 

结果

因原代滋养细胞和 HTR-8/ Svneo细胞两种细胞的实验结果完全一致,故将两种细胞合并用“滋养细胞”表述。

第一部分:

在LCHAD基因转录起始位点上游2000bp区域内的17个CpG位点中获得11     

   个位点(65%)的甲基化程度,其位置分别为-984、-960、-899、-853、-811、

   -796、-774、-727、-615、-595、-579位点,其中-899、-796及-774为复

   合位点(含有2个及以上CG序列),另外8个为单独位点(含有1个CG序

列),在不同研究组中每个位点的甲基化变化程度各不同,其中-899位点变

化最明显。

LCHAD基因启动子区CpG岛甲基化程度变化分析:(1)长、中链游离脂肪酸

 

 

对LCHAD基因启动子区CpG岛甲基化程度影响伴随时间的延长呈现升高趋势:

LC-FFA组孵育72h(0.55±0.08)比孵育48h(0.35±0.12)及24h(0.31±0.04)均明显升高(P<0.05),48h虽比24h有升高,但差异无统计学意

义(P>0.05)。MC-FFA组孵育72h(0.44±0.05)比孵育24h(0.31±0.04)明显升高(P<0.05)。(2)不同链长游离脂肪酸在不同作用时间影响LCHAD

基因启动子区CpG岛甲基化程度比较:LC-FFA组孵育72h 的甲基化程度比MC-FFA、SC-FFA组及F-FFA组的72、48及24h均明显升高(P<0.05)。

作用72h游离脂肪酸对LCHAD基因启动子区11个CpG位点甲基化程度影响

比较:在LC-FFA组和MC-FFA组-899位点甲基化程度明显高于其它位点(P

 <0.05)。

长链游离脂肪酸对LCHAD基因启动子区-899位点甲基化程度影响伴随时间

的延长呈现升高趋势(P<0.05)。LC-FFA组孵育72h比MC-FFA、SC-FFA

组及F-FFA组的72、48及24h均明显升高(P<0.05)。

第二部分:

病理妊娠组滋养细胞中甲基化修饰较高程度的位点为-899、-853、-615及

   -595位点,子痫前期组、HELLP 组-899、-853 及-615 CG 位点的甲基化程

   度均明显高于对照组(P<0.01),子痫前期组-853位点的甲基化程度明显高

   于HELLP组(P<0.05);HELLP组、APS组及对照组-595位点均呈无甲基化

   状态,分别与子痫前期组比较,差异均有统计学意义(P<0.01)。

子痫前期组滋养细胞中 LCHAD mRNA的表达水平为0.048±0.005,HELLP组

   为0.045±0.006,APS组为0.044±0.004,均显著低于对照组的 0.076 ±

   0.009(P<0.01);

子痫前期组滋养细胞中LCHAD 基因启动子区-899、-853、-727、-615及-579

   CG位点的甲基化程度与基因mRNA的表达水平均呈明显负相关(P<0.05);

   HELLP 组滋养细胞中 LCHAD 基因启动子区-899、-853、-615位点的甲基化

   程度与基因 mRNA的表达水平均呈明显负相关(P<0.05);而APS和对照组

   滋养细胞中两者均无显著相关性(P>0.05)。

 

 

 

结论

1. 长链游离脂肪酸比中链和短链游离脂肪酸呈现较大的滋养细胞LCHAD基因启动子区甲基化的修饰作用,并且伴随作用时间延长表现出明显的时间依赖效应。

2. 滋养细胞LCHAD基因启动子区CpG岛内各CG位点在长链脂肪酸作用下甲基化水平变化不一致,甲基化程度改变主要发生在LCHAD基因启动子区-899位点。

3. 早发重度子痫前期、HELLP综合征、APS对滋养细胞LCHAD基因启动子区的甲基化修饰存在差异,早发重度子痫前期、HELLP综合征滋养细胞中LCHAD基因启动子区甲基化修饰程度明显升高。

4. 早发重度子痫前期、HELLP综合征滋养细胞中LCHAD基因启动子区甲基化修饰程度与LCHAD mRNA表达水平呈明显负相关性。

英文摘要:

Objective

1.To explore the DNA methylation level of long- chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) gene promoter region in mitochondria of trophoblasts incubated with long-chain fatty acids and the time-effect of methylation.

2.By detecting the DNA methylation and gene expression of long- chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) in trophoblast cells, analyze the correlation of DNA methylation and gene expression in early-onset preeclampsia (EPE), hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome and antiphospholipid syndrome (APS), to investigate the molecular basis of long-chain fatty acid oxidation changes in different  preeclampsia and pathological pregnancy.

Methods

1.Primary human trophoblast cells and HTR8/Svneo cells were incubated

  with free fatty acids of various lengths. Long-chain free fatty acids

  (LC-FFA) was experimental group, short-chain fatty acids (SC-FFA) and

 

 

   medium-chain fatty acids (MC-FFA) were control groups, and blank

   control was without free fatty acid. Collecting cells and extract DNA

   at 24, 48 and 72h incubation respectively. Predicted CpG island

   location access to 2000bp DNA sequences upstream of the tranion

   start site of LCHAD gene. We designed methylation detection sites and

 primer originally. Methylation of CpG sites in LCHAD gene promoter

   region were detected by MassARRAY and analyzed statistically.

2. Primary human cytotrophoblast cells and HTR8/SVneo cells were treated with serum from patients with EPE (14 cases), HELLP (12 cases), APS (14 cases), and normal pregnant women (NP, 14 cases). The methylation level of LCHAD gene promoter region through the MassARRAY platform and mRNA expression level by real-time fluorescent quantitative PCR technique were conducted.

Results

First Part:

1. Cytosine-phosphate-guanine(CpG)sites in human LCHAD DNA promoter region: CpG sites were detected in the range of 558 bp before LCHAD gene tranion start site, the detected CpG sites were 11 sites including 8 single sites and 3 complex sites. The position of these sites were at -984, -960, -899, -853, -811, -796, -774, -727, -615,-595, -579 respectively.

2. Methylation of CpG island in LCHAD gene promoter region: (1)Methylation of CpG island in LC-FFA and MC-FFA groups showed rising trend with the time: Methylation level of LC-FFA group at 72h(0.55±0.08)was significantly higher than that of 48h (0.35±0.12)and 24h(0.31±0.04)(P<0.05). Methylation level of MC-FFA group at 72h(0.44±0.05)was significantly higher than that of 24h(0.31±0.04)(P<0.05).(2)

 

 

   Methylation of CpG island in LCHAD gene promoter region in different groups at different times: Methylation level of LC-FFA group at 72h

   was significantly higher than that of other  three groups at 72,48 and 24h(P<0.05).

3. Methylation of 11 sites in LCHAD gene promoter region in different

 groups at 72h: Methylation level of -899 site in LC-FFA and MC-FFA

   groups were significantly higher than that of other sites(P<0.05).

Methylation level of -899 site in LCHAD gene promoter region in LC-FFA

group showed rising trend with the time: [72h(0.34±0.15),48h

  (0.14±0.05)and 24h(0.10±0.02), P<0.05]. Methylation level of

   LC-FFA group at 72h was significantly higher than that of other three

   groups at 72,48 and 24h(P<0.05).

Second Part

 1. The sites of -899, -853, -615 and -595 showed increased methylation level in EPE and HELLP groups. The methylation level at -899, -853 and -615 sites in EPE and HELLP groups were significantly higher than in NP group(P<0.01). The methylation level at -853 site was higher in EPE group than that in HELLP group(P<0.05). The -595 site showed unmethylated in EPE, HELLP and APS groups. There were significantly difference between the 3 groups and EPE group(P<0.01).

 2. The gene expression of LCHAD mRNA in EPE(0.048±0.005), HELLP

   (0.045±0.006) and APS (0.044±0.004) groups were significantly

 lower than NP group(0.076±0.009;P<0.01).

 3. The correlation of methylation level and gene expression in all

    groups:

    the methylation level at -899, -853, -727, -615 and -579 sites were

    negatively correlated with gene mRNA expression in EPE group(P<

 

 

    0.05). The methylation level at -899, -853 and -615 sites were

    negatively correlated with gene mRNA expression in HELLP group(P<0.05).

 

Conclusions

Methylation modification effect on LCHAD gene promoter region in

trophoblast cells incubated with long-chain fatty acids is more

significant than with medium-chain and short-chain fatty acids. Methylation modification effect on LCHAD gene promoter region in

trophoblast cells incubated with long-chain fatty acids showed obvious time-effect as incubation time prolonged.

2. The changes at - 899 site dominate the degree of methylation in LCHAD gene promoter region.

3. Methylation modification effect on LCHAD gene promoter region in early-onset preeclampsia and HELLP syndrome groups were more significant than APS group.

4. The different correlation of LCHAD DNA methylation and gene expression in different pathological groups. LCHAD DNA methylation of early-onset preeclampsia and HELLP syndrome were negativly correlated with LCHAD gene expression.

语种: 中文
相关网址: 查看原文
内容类型: 学位论文
URI标识: http://ir.bjmu.edu.cn/handle/400002259/124744
Appears in Collections:北京大学第三临床医学院_学位论文

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作者单位: 北京大学第三临床医学院

Recommended Citation:
孟然. 子痫前期滋养细胞线粒体LCHAD基因甲基化与基因表达相关性的[D]. 北京大学第三临床医学院. 北京大学. 2016.
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