本文選題：前期 + 胎盤�。� 參考：《山東大學(xué)》2014年博士論文

【摘要】：一、應(yīng)用cDNA基因芯片篩選子癇前期胎盤與正常胎盤差異表達基因： 研究背景：子癇前期-子癇(preeclampsia-eclampsia)是妊娠期特發(fā)性疾病,病因和發(fā)病機制至今仍未完全闡明,是產(chǎn)科重要的研究課題。大量研究結(jié)果提示子癇前期的發(fā)生機制可能為多基因的遺傳學(xué)因素與環(huán)境因素交互作用的結(jié)果所致。胎盤異常在子癇前期的發(fā)病中起著重要作用。隨著人類基因組研究的深入,易感基因成為子癇前期遺傳學(xué)研究的熱點。 材料和方法：選取2012年8月于山東省立醫(yī)院產(chǎn)科剖宮產(chǎn)分娩的年齡、產(chǎn)次相匹配的重度子癇前期及正常孕婦胎盤組織標(biāo)本各10例。抽提和處理胎盤組織RNA; cDNA第一鏈制備探針,熒光探針純化并定量；制備微陣列,采用上海聯(lián)合基因公司的的BIOSTARH140s基因芯片(cDNA表達譜芯片),芯片雜交、洗滌；采用ScanArray4000掃描儀(Packard Biochip Technologies, Inc. USA)掃描芯片,獲得實驗數(shù)據(jù),進行數(shù)據(jù)歸一化處理及芯片掃描質(zhì)控。 基因芯片雜交結(jié)果：cDNA表達譜芯片雜交結(jié)果顯示,正反雙向標(biāo)記子癇前期患者和正常對照樣本,熒光交換處理可避免熒光非特異性吸附所造成的假陽性率,結(jié)果顯示第一次實驗結(jié)果和互換熒光標(biāo)記后的第二次實驗結(jié)果分別得到382個和394個差異表達基因,兩次實驗共同的差異基因為111個,百分率分別為29.1%和28.2%。在111個表達差異的基因中,在子癇前期組中呈上調(diào)表達(比值≥2.0)的基因有68個,呈下調(diào)表達(比值≤0.5)的基因有44個。經(jīng)MatchMiner軟件處理后,獲取GENEBANK ID。 二、cDNA基因芯片子癇前期胎盤組織差異表達基因生物信息學(xué)分析 方法：使用DAVID (The Database for Annotation, Visualization andlntegrated Discovery)分析工具對cDNA基因芯片獲取的子癇前期胎盤組織差異表達基因進行基因本體論(gene ontology, GO)分析和富集分析。 結(jié)果：功能聚類工具獲取兩組富集分?jǐn)?shù)大于2的基因簇。功能注釋功能注釋工具分別進行以下分析：功能注釋圖(Functional Annotation Chart)分析結(jié)果：共有86個顯著性功能G0分類項,包含715個差異基因。11個GO terms中(FDR0.05)的基因在分子功能中,與結(jié)構(gòu)分子活性、催化活性相關(guān)；在生物學(xué)過程中,與生物黏附、細胞過程、代謝過程、結(jié)構(gòu)區(qū)域構(gòu)建、結(jié)構(gòu)區(qū)域相關(guān)；在細胞組分中,與細胞外區(qū)域及部分細胞外區(qū)域相關(guān)。功能注釋聚類(Functional Annotation Clustering),和功能注釋圖一樣,指向糖蛋白、信號、信號肽、雙硫鍵、細胞外區(qū)域及部分細胞外區(qū)域。功能注釋表(Functional Annotation Table)對輸入基因逐一做出功能注釋。最后篩選出與子癇前期關(guān)系最為密切的16種目的基因。 三、應(yīng)用實時熒光定量PCR方法驗證子癇前期胎盤組織差異表達基因 理論基礎(chǔ)：Real-time PCR原理是在PCR反應(yīng)體系中加入熒光基團,利用檢測熒光信號累積實時監(jiān)測PCR進程,對PCR的全部反應(yīng)擴增過程進行實時的監(jiān)測,連續(xù)的分析與擴增相關(guān)聯(lián)的熒光信號,監(jiān)測到的熒光信號隨反應(yīng)時間的變化可以繪制成一條曲線。最后通過標(biāo)準(zhǔn)曲線或數(shù)學(xué)公式對未知模板進行定量分析,通過標(biāo)準(zhǔn)曲線或數(shù)學(xué)公式計算出樣品的起始拷貝數(shù)。 材料與方法：對篩選出了16種子癇前期關(guān)鍵候選基因應(yīng)用Real-time PCR方法觀察其在mRNA水平上的變化。臨床資料：同前。提取胎盤組織總RNA,合成引物,按照RNA反轉(zhuǎn)錄試劑盒提供的反應(yīng)體系進行反轉(zhuǎn)錄反應(yīng)。定量PCR反應(yīng),生成標(biāo)準(zhǔn)曲線。 實驗結(jié)果：Real Time-PCR檢測子癇前期胎盤組織與正常胎盤組織的的表達差異,其中有3個降調(diào),13個上調(diào),與基因芯片的結(jié)果對比,兩種方法有很好的相關(guān)性,16個基因不僅變化方向一致,而且基因表達水平很接近。 四、結(jié)果分析與討論： 經(jīng)生物信息學(xué)分析篩選出子癇前期胎盤組織中發(fā)揮重要作用的差異表達基因,主要包括：(1)滋養(yǎng)細胞浸潤相關(guān)因子：潛在性轉(zhuǎn)移生長因子β結(jié)合蛋白2(latent transforming growth factor βbinding protein2,NM_000428)胰島素樣生長因子結(jié)合蛋白1(CR595377)等；(2)免疫相關(guān)因子：干擾素受體1(NM_000629)甘露糖受體Cl(NM_002438)等；(3)妊娠相關(guān)蛋白等(AF342989、 M23575、BC063127)等；(4)細胞信號通路相關(guān)基因：(AK129833、BC012609)等；(5)其它：細胞角蛋白類(AK122864)、脆性X綜合征基因(BC067272)細胞色素P450(NM031226)等。 對結(jié)果進行分析討論,得出以下結(jié)論： 1、子癇前期患者胎盤組織的基因表達與正常胎盤組織存在著很大的差異,表明子癇前期患者胎盤存在代謝失衡。 2、基因芯片技術(shù)是篩查子癇前期胎盤組織差異表達基因高效、快捷的工具,實時熒光定量RT-PCR可以作為單個基因表達變化的研究,兩者互為補充和驗證。 3、基因功能富集分析是得出與實驗?zāi)康挠酗@著關(guān)聯(lián)的、低假陽性率的及靶向性的基因功能類別的重要手段。 4、在子癇前期胎盤組織一些基因表達異常,表明其可能參與了子癇前期的發(fā)病機制,也可能是子癇前期病理生理變化的結(jié)果。 5、潛在性轉(zhuǎn)移生長因子β結(jié)合蛋白2(LTBP-2)表達異常降低,抑制滋養(yǎng)細胞的浸潤,螺旋動脈重鑄不完全,引起胎盤缺血缺氧,繼而激活和損傷血管內(nèi)皮細胞,促進子癇前期一子癇的發(fā)生。 6、胰島素樣生長因子結(jié)合蛋白1(IGFBP1)表達異常升高,導(dǎo)致滋養(yǎng)細胞侵蝕障礙,胎盤淺著床,從而引發(fā)子癇前期 7、妊娠特異性蛋白包括妊娠相關(guān)血漿蛋白A (PAPP-A)、妊娠相關(guān)血漿蛋白B(PAPP-B)及妊娠相關(guān)血漿蛋白C(PAPP-C)即妊娠特異性β1糖蛋白(PSG),的表達異常升高,抑制母體排異、延長妊娠時間、保證胎兒存活。 8、甘露糖受體家族基因(mannose receptor gene1,MRC1)在S期調(diào)節(jié)復(fù)制叉聚合分離,影響DNA復(fù)制,干擾基因的遺傳穩(wěn)定性。 本研究通過基因芯片技術(shù)對子癇前期胎盤組織差異表達基因進行研究,發(fā)現(xiàn)111條有差異表達的基因,通過DAVID軟件進行生物信息學(xué)分析,篩選出16種子癇前期胎盤組織中發(fā)揮重要作用的基因,并進行實時熒光定量RT-PCR驗證,為尋找子癇前期胎盤相關(guān)基因提供線索。但子癇前期發(fā)病和影響因素復(fù)雜,尤其是重度子癇前期患者臨床表現(xiàn)多樣化,各病例之間存在個體差異,確定差異表達基因的意義及研究各基因間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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2014
【分類號】：R714.244

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