子痫前期胎盘组织差异性基因表达及生物信息学分析

发布时间：2018-04-18 06:14

本文选题：前期 + 胎盘　；参考：《山东大学》2014年博士论文

【摘要】：一、应用cDNA基因芯片筛选子痫前期胎盘与正常胎盘差异表达基因：研究背景：子痫前期-子痫(preeclampsia-eclampsia)是妊娠期特发性疾病,病因和发病机制至今仍未完全阐明,是产科重要的研究课题。大量研究结果提示子痫前期的发生机制可能为多基因的遗传学因素与环境因素交互作用的结果所致。胎盘异常在子痫前期的发病中起着重要作用。随着人类基因组研究的深入,易感基因成为子痫前期遗传学研究的热点。材料和方法：选取2012年8月于山东省立医院产科剖宫产分娩的年龄、产次相匹配的重度子痫前期及正常孕妇胎盘组织标本各10例。抽提和处理胎盘组织RNA; cDNA第一链制备探针,荧光探针纯化并定量；制备微阵列,采用上海联合基因公司的的BIOSTARH140s基因芯片(cDNA表达谱芯片),芯片杂交、洗涤；采用ScanArray4000扫描仪(Packard Biochip Technologies, Inc. USA)扫描芯片,获得实验数据,进行数据归一化处理及芯片扫描质控。基因芯片杂交结果：cDNA表达谱芯片杂交结果显示,正反双向标记子痫前期患者和正常对照样本,荧光交换处理可避免荧光非特异性吸附所造成的假阳性率,结果显示第一次实验结果和互换荧光标记后的第二次实验结果分别得到382个和394个差异表达基因,两次实验共同的差异基因为111个,百分率分别为29.1%和28.2%。在111个表达差异的基因中,在子痫前期组中呈上调表达(比值≥2.0)的基因有68个,呈下调表达(比值≤0.5)的基因有44个。经MatchMiner软件处理后,获取GENEBANK ID。二、cDNA基因芯片子痫前期胎盘组织差异表达基因生物信息学分析方法：使用DAVID (The Database for Annotation, Visualization andlntegrated Discovery)分析工具对cDNA基因芯片获取的子痫前期胎盘组织差异表达基因进行基因本体论(gene ontology, GO)分析和富集分析。结果：功能聚类工具获取两组富集分数大于2的基因簇。功能注释功能注释工具分别进行以下分析：功能注释图(Functional Annotation Chart)分析结果：共有86个显著性功能G0分类项,包含715个差异基因。11个GO terms中(FDR0.05)的基因在分子功能中,与结构分子活性、催化活性相关；在生物学过程中,与生物黏附、细胞过程、代谢过程、结构区域构建、结构区域相关；在细胞组分中,与细胞外区域及部分细胞外区域相关。功能注释聚类(Functional Annotation Clustering),和功能注释图一样,指向糖蛋白、信号、信号肽、双硫键、细胞外区域及部分细胞外区域。功能注释表(Functional Annotation Table)对输入基因逐一做出功能注释。最后筛选出与子痫前期关系最为密切的16种目的基因。三、应用实时荧光定量PCR方法验证子痫前期胎盘组织差异表达基因理论基础：Real-time PCR原理是在PCR反应体系中加入荧光基团,利用检测荧光信号累积实时监测PCR进程,对PCR的全部反应扩增过程进行实时的监测,连续的分析与扩增相关联的荧光信号,监测到的荧光信号随反应时间的变化可以绘制成一条曲线。最后通过标准曲线或数学公式对未知模板进行定量分析,通过标准曲线或数学公式计算出样品的起始拷贝数。材料与方法：对筛选出了16种子痫前期关键候选基因应用Real-time PCR方法观察其在mRNA水平上的变化。临床资料：同前。提取胎盘组织总RNA,合成引物,按照RNA反转录试剂盒提供的反应体系进行反转录反应。定量PCR反应,生成标准曲线。实验结果：Real Time-PCR检测子痫前期胎盘组织与正常胎盘组织的的表达差异,其中有3个降调,13个上调,与基因芯片的结果对比,两种方法有很好的相关性,16个基因不仅变化方向一致,而且基因表达水平很接近。四、结果分析与讨论：经生物信息学分析筛选出子痫前期胎盘组织中发挥重要作用的差异表达基因,主要包括：(1)滋养细胞浸润相关因子：潜在性转移生长因子β结合蛋白2(latent transforming growth factor βbinding protein2,NM_000428)胰岛素样生长因子结合蛋白1(CR595377)等；(2)免疫相关因子：干扰素受体1(NM_000629)甘露糖受体Cl(NM_002438)等；(3)妊娠相关蛋白等(AF342989、 M23575、BC063127)等；(4)细胞信号通路相关基因：(AK129833、BC012609)等；(5)其它：细胞角蛋白类(AK122864)、脆性X综合征基因(BC067272)细胞色素P450(NM031226)等。对结果进行分析讨论,得出以下结论： 1、子痫前期患者胎盘组织的基因表达与正常胎盘组织存在着很大的差异,表明子痫前期患者胎盘存在代谢失衡。 2、基因芯片技术是筛查子痫前期胎盘组织差异表达基因高效、快捷的工具,实时荧光定量RT-PCR可以作为单个基因表达变化的研究,两者互为补充和验证。 3、基因功能富集分析是得出与实验目的有显著关联的、低假阳性率的及靶向性的基因功能类别的重要手段。 4、在子痫前期胎盘组织一些基因表达异常,表明其可能参与了子痫前期的发病机制,也可能是子痫前期病理生理变化的结果。 5、潜在性转移生长因子β结合蛋白2(LTBP-2)表达异常降低,抑制滋养细胞的浸润,螺旋动脉重铸不完全,引起胎盘缺血缺氧,继而激活和损伤血管内皮细胞,促进子痫前期一子痫的发生。 6、胰岛素样生长因子结合蛋白1(IGFBP1)表达异常升高,导致滋养细胞侵蚀障碍,胎盘浅着床,从而引发子痫前期 7、妊娠特异性蛋白包括妊娠相关血浆蛋白A (PAPP-A)、妊娠相关血浆蛋白B(PAPP-B)及妊娠相关血浆蛋白C(PAPP-C)即妊娠特异性β1糖蛋白(PSG),的表达异常升高,抑制母体排异、延长妊娠时间、保证胎儿存活。 8、甘露糖受体家族基因(mannose receptor gene1,MRC1)在S期调节复制叉聚合分离,影响DNA复制,干扰基因的遗传稳定性。本研究通过基因芯片技术对子痫前期胎盘组织差异表达基因进行研究,发现111条有差异表达的基因,通过DAVID软件进行生物信息学分析,筛选出16种子痫前期胎盘组织中发挥重要作用的基因,并进行实时荧光定量RT-PCR验证,为寻找子痫前期胎盘相关基因提供线索。但子痫前期发病和影响因素复杂,尤其是重度子痫前期患者临床表现多样化,各病例之间存在个体差异,确定差异表达基因的意义及研究各基因间的可能存在的相互作用仍有有待于更全面、深入的研究。
[Abstract]:First, cDNA gene chip was used to screen the differentially expressed genes in preeclampsia placenta and normal placenta.
Background: pre eclampsia (preeclampsia-eclampsia) is a pregnancy idiopathic disease, etiology and pathogenesis has not been fully elucidated, is an important research topic in obstetrics. A large number of research results of genetics possible pathogenesis of preeclampsia is that multiple genes for interaction elements and environmental factors in the pathogenesis of placental abnormality caused by. Preeclampsia plays an important role. With the human genome research, genetic susceptibility has become the focus of the genetic study on preeclampsia.
Materials and methods: from August 2012 to the Shangdong Province-owned Hospital obstetric cesarean delivery age, parity, severe preeclampsia and normal placenta samples from 10 patients. Treatment of placenta extract and RNA; the first strand cDNA probe preparation, purification and quantitative fluorescence probe; microarray preparation, the Shanghai United gene company BIOSTARH140s gene chip (microarray cDNA), hybridization, washing; using a ScanArray4000 scanner (Packard Biochip Technologies, Inc. USA) chip, experimental data, data normalization and chip scanning quality control.
The results of gene chip hybridization: microarray hybridization results showed that cDNA, bidirectional markers of preeclampsia patients and normal control samples, fluorescence exchange treatment can avoid the false positive rate of fluorescence caused by nonspecific adsorption, results show that the results of the first experiment and second test results after swap fluorescence labeling were obtained 382 and 394 difference the expression of genes, gene two experiment common difference is 111, the percentage is 29.1% and 28.2%. respectively in the 111 differentially expressed genes, in preeclampsia group was upregulated (ratio = 2) based on 68, was down regulated (ratio of less than 0.5) of the 44 genes. With MatchMiner software, GENEBANK ID.
Two, bioinformatics analysis of differential expression gene of placental tissue in preeclampsia of cDNA gene chip
Methods: DAVID (The Database for Annotation, Visualization andlntegrated Discovery) analysis tool was used to analyze the differentially expressed genes in placenta tissues of preeclampsia by cDNA gene chip.
Results: the function of clustering tool to obtain two enrichment scores more than 2 gene clusters. Functional annotation functional annotation tools are the following analysis: functional annotation diagram (Functional Annotation Chart) analysis results: there are 86 significant features of G0 classification, including 715.11 GO terms gene (FDR0.05) gene in molecular function, activity and molecular structure, catalytic activity; in the biological process, and biological adhesion, cell process, metabolic process, construction of regional structure, regional structure; in the cell fractions, and the extracellular domain and extracellular domain. Functional annotation clustering (Functional Annotation Clustering),, to map and functional annotation of glycoprotein, signal, signal peptide, disulfide bond, extracellular domain and extracellular domain. The functional annotation table (Functional Annotation Table) of input genes one by one In the end, 16 kinds of target genes, which are most closely related to preeclampsia, are selected.
Three, the application of real time fluorescence quantitative PCR to verify the differential expression gene of placental tissue in preeclampsia
Theoretical basis: Real-time PCR principle is adding fluorophore in PCR reaction system, the cumulative process real-time monitoring of PCR detected by fluorescence signals of all reactions of PCR amplification process real-time monitoring, the fluorescence signal associated with the amplification analysis of continuous change, the fluorescence signal detected with reaction time can be plotted as a curve. Finally, quantitative analysis of the unknown template by standard curve or mathematical formula, by standard curve or mathematical formula to calculate the initial copy number of samples.
Materials and methods: We selected 16 seed preeclampaia key candidate gene using Real-time PCR method to observe the changes in the level of mRNA. Clinical data: ibid. Total RNA was extracted from the placenta of the primer reaction system provides RNA reverse transcription kit according to the reverse transcription reaction. Quantitative PCR reaction, generate standard curve.
Results: the expression of Real Time-PCR detected in preeclampsia placenta and normal placenta, which has 3 flats, 13 increase, compared with the microarray results, there is a good correlation between the two methods, 16 genes not only change the direction, and the gene expression level is very close.
Four, result analysis and discussion:
By bioinformatics analysis, screening of preeclampsia placenta plays an important role in the differences of gene expression, including: (1) trophoblast invasion related factors: latent transforming growth factor binding protein 2 (latent transforming growth factor protein2 beta binding, NM_000428) insulin-like growth factor binding protein 1 (CR595377); (2) immune related factors: interferon receptor 1 (NM_000629) of mannose receptor Cl (NM_002438); (3) pregnancy related proteins (AF342989, M23575, BC063127); (4) cell signal transduction genes (AK129833, BC012609); (5) other: cyokeratin (AK122864), fragile X syndrome gene (BC067272) and cytochrome P450 (NM031226).
The results are analyzed and discussed, and the following conclusions are drawn:
1, there are significant differences with normal expression in placenta of preeclampsia placenta gene show preeclampsia placenta were metabolic imbalance.
2, gene chip technology is an effective and quick tool to screen differentially expressed genes in placenta tissues of preeclampsia. Real-time fluorescence quantitative RT-PCR can be used as a single gene expression change research, both of which complement each other and verify.
3, gene function enrichment analysis is an important means to draw a significant association with experimental purposes, with low false positive rates and targeted gene functional categories.
4, abnormal expression of some genes in placenta tissues during preeclampsia suggests that it may be involved in the pathogenesis of preeclampsia, or it may be the result of pathophysiological changes in preeclampsia.
5, the potential transfer growth factor beta binding protein 2 (LTBP-2) expression decreased abnormally, inhibited the infiltration of trophoblast cells, incomplete recanking of spiral arteries, caused placental ischemia and hypoxia, then activated and damaged vascular endothelial cells, and promoted the occurrence of eclampsia in preeclampsia.
6, insulin-like growth factor binding protein 1 (IGFBP1) expression of abnormally elevated trophoblast cells leads to the erosion barriers, shallow placental implantation, which lead to pre eclampsia
7, pregnancy specific protein, including pregnancy related plasma protein A (PAPP-A), pregnancy related plasma protein B (PAPP-B) and pregnancy related plasma protein C (PAPP-C), namely pregnancy specific beta 1 glycoprotein (PSG), increased significantly, inhibited maternal rejection, prolonged pregnancy time, and ensured fetal survival.
8, mannose receptor gene1 (MRC1) regulates and separates the replication fork polymerization in S stage, which affects the replication of DNA and interferes with the genetic stability of the gene.
This study through the research on the differences of gene microarray technology in preeclampsia placental tissue expression, found 111 differentially expressed genes, bioinformatics analysis by DAVID software, selected 16 genes play an important role in seed preeclampsia placenta, and real-time quantitative RT-PCR validation, provide clues for preeclampsia the placenta related genes. But the pathogenesis of preeclampsia and the influence of complex factors, especially in patients with severe preeclampsia diverse clinical manifestations, there are individual differences among patients and determine the expression difference of interaction between meaning and research of each gene between genes may still need to be more comprehensive and in-depth research.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R714.244

【共引文献】