【摘要】：研究背景及目的子宮腺肌病是女性常見疾病,主要臨床表現(xiàn)為不同程度的繼發(fā)性進行性痛經(jīng)、異常子宮出血,可能導(dǎo)致不孕不育和早期流產(chǎn)等,嚴(yán)重影響女性的身心健康和生活質(zhì)量。至今為止其發(fā)病機制仍不清楚。目前關(guān)于其發(fā)病機制存在多種學(xué)說,最廣為接受的學(xué)說認(rèn)為子宮內(nèi)膜內(nèi)陷于子宮肌層并異位增殖導(dǎo)致該病的發(fā)生。研究發(fā)現(xiàn),該病的在位內(nèi)膜存在多種異常,涉及遺傳、激素、免疫和代謝等多個方面。但是編碼基因相關(guān)的研究較為零散,通過組學(xué)的方法系統(tǒng)篩選該病在位內(nèi)膜編碼基因的改變有助于揭示該病的發(fā)生機制。除了編碼基因的改變以外,也存在表觀遺傳的異常變化。lncRNAs作為功能調(diào)節(jié)元件在基因表達調(diào)控中發(fā)揮重要的作用,并參與多種重要信號通路的調(diào)節(jié)。其異常表達與多種良惡性疾病相關(guān)。lncRNAs在子宮腺肌病發(fā)生發(fā)展中的作用尚無研究報道。本研究的目的為：1. 構(gòu)建子宮腺肌病在位內(nèi)膜與對照內(nèi)膜lncRNAs和nRNAs的差異表達譜；2. 對lncRNA和mRNA的功能進行生物學(xué)信息分析,以探討其可能的調(diào)控機制并得出潛在的核心調(diào)節(jié)基因；3. 對部分潛在核心調(diào)節(jié)基因的表達水平進行檢測,為后續(xù)功能實驗提供研究基礎(chǔ)。研究方法1. 利用基因芯片技術(shù)檢測子宮腺肌病在位內(nèi)膜和對照內(nèi)膜組織中l(wèi)ncRNAs和mRNAs的表達,構(gòu)建lncRNAs和mRNAs差異表達譜(其中兩組的樣本量分別為4例)。2. 利用實時熒光定量PCR技術(shù)驗證芯片結(jié)果中部分差異表達IncRNAs(共6個),以進一步驗證基因芯片實驗結(jié)果。3. 利用生物信息學(xué)手段對差異表達的lncRNAs和mRNAs進行分析。3.1 GO分析：對芯片結(jié)果中差異mRNAs進行顯著性功能分析,得到差異基因參與的具有顯著性、低誤判率、靶向性的功能。3.2 Pathway分析：將差異nRNAs進行Pathway顯著性分析,得到差異基因參與的具有顯著性、低誤判率、靶向性的Pathway。3.3構(gòu)建共表達網(wǎng)絡(luò)：通過實驗組與對照組比較得到的差異mRNAs、差異lncRNAs在芯片中的實測表達值,分別構(gòu)建實驗組與對照組的共表達網(wǎng)絡(luò),通過比較共表達網(wǎng)絡(luò)上的差異來定位兩組樣本中的潛在核心調(diào)節(jié)基因。4. 利用實時熒光定量PCR和免疫組化法檢測部分潛在核心調(diào)節(jié)基因——TLN1和CCND2在子宮腺肌病在位內(nèi)膜中的表達。研究結(jié)果1. 子宮腺肌病在位內(nèi)膜與對照內(nèi)膜相比,存在165個差異表達lncRNAs和61 2個差異表達mRNAs。在差異表達的lncRNAs中有117個lncRNAs表達下調(diào)和48個lncRNAs表達上調(diào)。其中下調(diào)最明顯的lncRNA為n333955(差異倍數(shù)為：0.0032),上調(diào)最明顯的lncRNA為n342839(差異倍數(shù)為4.13)。在差異表達的mRNAs中,下調(diào)的nRNAs,總數(shù)為414個,上調(diào)的mRNAs總數(shù)198個。其中下調(diào)最明顯的mRNA為HBA1(差異倍數(shù)為：0.02),上調(diào)最明顯的mRNA為THBS2(差異倍數(shù)為5.14)。2. 選擇6個lncRNAs進行實時熒光定量PCR驗證,結(jié)果顯示n333955、n337373、n338909這3個lncRNAs在子宮腺肌病在位內(nèi)膜組織中表達水平下降,而n341651、n342794、n387706表達水平升高,差異均具有統(tǒng)計學(xué)意義,與芯片檢測的表達趨勢相符。3. 生物學(xué)信息分析結(jié)果：3.1通過顯著靶向性GO分析,得出上調(diào)差異基因參與的顯著性功能共352項,包括：細(xì)胞外基質(zhì)組成、內(nèi)皮細(xì)胞分化、細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)、細(xì)胞遷移的正調(diào)控、血管生成等。下調(diào)差異基因參與的顯著性功能共283項,包括：基因表達、DNA復(fù)制、細(xì)胞分裂、有絲分裂細(xì)胞周期的G1/S期過渡、免疫反應(yīng)等。3.2通過顯著性Pathway分析,得到上調(diào)差異基因參與的顯著性信號轉(zhuǎn)導(dǎo)通路共40項,包括MAPK信號通路、局部粘附、粘附連接、細(xì)胞外基質(zhì)受體相互作用、PI3K-Akt信號通路等。下調(diào)差異基因參與的顯著性信號轉(zhuǎn)導(dǎo)通路共39項,包括：DNA復(fù)制、細(xì)胞周期、原發(fā)性免疫缺陷、細(xì)胞因子、細(xì)胞因子受體相互作用等。3.3分別構(gòu)建了兩組樣本的共表達網(wǎng)絡(luò),兩張共表達網(wǎng)絡(luò)的結(jié)構(gòu)存在明顯差別。根據(jù)基因在兩個網(wǎng)絡(luò)中共表達地位的變化情況,篩選出對樣本差異起重要作用的關(guān)鍵mRNAs/lncRNAs,包括TLN1、n342794、MYBL1、CCND2、 ENST00000406939、n338909等4. 實時熒光定量PCR和免疫組化結(jié)果顯示,TLN1基因和蛋白在子宮腺肌病在位內(nèi)膜組織中的表達明顯高于對照內(nèi)膜組織；CCND2基因和蛋白在子宮腺肌病在位內(nèi)膜組織中的表達明顯低于對照內(nèi)膜組織,差異均具有統(tǒng)計學(xué)意義。研究結(jié)論1. 首次通過芯片檢測成功構(gòu)建子宮腺肌病在位內(nèi)膜與對照內(nèi)膜lncRNAs和mRNAs的差異表達譜。2. 實時熒光定量PCR結(jié)果與芯片結(jié)果有很好的一致性,進一步驗證芯片結(jié)果的可信度。3. 通過生物信息學(xué)分析可初步探討差異mRNAs和lncRNAs在子宮腺肌病發(fā)生過程中可能的調(diào)控作用。4. 潛在核心調(diào)節(jié)基因TLN1和CCND2基因和蛋白在子宮腺肌病在位內(nèi)膜組織中表達異常。
[Abstract]:The study background and objective uterine adenomyosis are female urinary tract diseases. The main clinical manifestations include secondary progressive dysmenorrhea, abnormal uterine bleeding, infertility and early abortion, which seriously affect women's physical and mental health and quality of life. Until now its pathogenesis remains unclear. There are many theories about the pathogenesis of the disease, and the most widely accepted theory holds that the endometriosis is trapped in the uterine muscle layer and ectopic proliferation leads to the occurrence of the disease. It has been found that there are a variety of abnormalities in the endometrium of the disease, which involve many aspects such as heredity, hormone, immunity and metabolism. But the research on coding gene is fragmented, and the method of group learning can screen the change of the coding gene of the disease in order to reveal the pathogenesis of the disease. In addition to the change in the coding gene, there is an abnormal change in apparent genetic. It plays an important role in the regulation of gene expression as a function regulating element, and participates in the regulation of various important signal paths. Its abnormal expression is associated with various benign and malignant diseases. There is no study in the development of adenomyosis. The purpose of this study is: 1. To construct a differential expression profile between the endometrium of the adenomyosis and the endometrium of the control endometrium; 2. Biological information analysis was carried out on the functions of cRNA and mRNA to investigate possible regulatory mechanisms and to develop potential core regulatory genes; 3. The expression level of some potential core regulatory genes was tested to provide a basis for follow-up function experiments. Study Method 1. Gene chip technique was used to detect the expression of Jurkat and mCD44v6 in endometrial and control endometrial tissues, and the differential expression profiles were constructed (the sample sizes of the two groups were 4 cases, respectively). The results of gene chip experiment were verified by using real-time fluorescence quantitative polymerase chain reaction (PCR) technique to verify partial differential expression in the chip. 3. 1GO analysis: Significant sexual function analysis was performed on the differences in the results of the chip, and the differentially expressed genes were found to have significance, low misjudgment rate and targeting function. A co-expression network with significance, low misjudgment rate and targeting property of differential gene participation is obtained, and a co-expression network of the experimental group and the control group is constructed by comparing the difference mT obtained in the experimental group and the control group, respectively constructing the co-expression network of the experimental group and the control group, potential core regulatory genes in both sets of samples were positioned by comparing differences on co-expression networks. 4. The expression of partial potential core regulatory genes, TLN1 and CCND2 in the endometrium of adenomyosis was detected by real-time fluorescence quantitative PCR and immunohistochemistry. Study results 1. There were 165 differences in the expression of CD44v6 and 61 2 differences in the expression of mCD44v6 in the endometrium of adenomyosis compared with the control endometrium. 117 of the differentially expressed CDcRNAs were down-regulated and 48 of them were upregulated. Among them, the most significant cRNA was n333955 (difference multiple: 0. 0032), and the most significant cRNA was n342839 (the difference was 4. 13). Of the differentially expressed mRNAs, the down-regulated nnn, the total number is 414, the total number of mbs up-regulated 198. Among them, the most significant mRNA was HBA1 (difference fold: 0. 02), and the most significant mRNA was THBS2 (the difference is 5. 14). The results showed that the expression level of n333955, n337373, n338909 was decreased in the endometrial tissue of adenomyosis, while n341651, n342794, n387706 expression levels were elevated, and the difference was statistically significant. The results of biological information analysis showed that there were 352 significant sexual functions including extracellular matrix composition, endothelial cell differentiation, intracellular signal transduction, positive regulation of cell migration, angiogenesis and so on. The remarkable sexual function of down-regulation differential gene participation was 283, including: gene expression, DNA replication, cell division, cell cycle G1/ S transition, immune response and so on. include MAPK signaling pathways, local adhesion, adhesion connections, extracellular matrix receptor interactions, extracellular matrix-Akt signaling pathways, and the like. There are 39 significant signal transduction pathways for down-regulation of differential gene participation, including DNA replication, cell cycle, primary immunodeficiency, cytokines, cytokine receptor interactions, and the like. 3. 3 constructs co-expression networks of two groups of samples, respectively, There are significant differences in the structure of the two co-expression networks. According to the change of the expression status of the genes in the two networks, the key mdr/ cGVHD, including TLN1, n342794, MYBL1, CCND2, ENST00000406939, n338909, etc., plays an important role in the sample difference. Real-time fluorescence quantitative PCR and immunohistochemistry showed that the expression of TLN1 gene and protein in endometrial tissue was significantly higher than that of control endometrial tissue, and the expression of CCND2 gene and protein in endometrial tissue was significantly lower than that of control endometrial tissue. The difference was statistically significant. Study conclusion 1. In this paper, the differential expression profiles of the endometrium of the adenomyosis and the control endometrium were successfully constructed by the chip detection. the real-time fluorescence quantitative PCR result has good consistency with the chip result, and further verifies the reliability of the chip result. By means of bioinformatic analysis, it is possible to preliminarily explore the possible regulative effect of mpr and mckcpr in the course of the occurrence of adenomyosis. The potential core regulatory genes TLN1 and CCND2 genes and proteins express abnormalities in the endometrial tissue of the adenomyosis.
【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：R711.71

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7 史精華;子宮腺肌病子宮內(nèi)膜生長極向與平滑肌離子通道關(guān)系的研究[D];北京協(xié)和醫(yī)學(xué)院;2011年

8 李娟;單核細(xì)胞趨化蛋白-1、基質(zhì)細(xì)胞衍生因子-1在子宮腺肌病中的表達及意義[D];山東大學(xué);2012年

9 任月芳;自噬相關(guān)基因Beclin 1在子宮腺肌病中的作用及缺氧對Beclin 1影響的研究[D];浙江大學(xué);2010年

10 楊艷峰;GPR30介導(dǎo)的雌激素非經(jīng)典途徑在子宮腺肌病發(fā)病機制中的研究[D];浙江大學(xué);2013年

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1 袁亞敏;子宮腺肌病的中西醫(yī)研究進展[D];成都中醫(yī)藥大學(xué);2007年

2 張婭;GNRH-a聯(lián)合LNG-IUS治療輕、中度子宮腺肌病臨床療效觀察[D];遵義醫(yī)學(xué)院;2015年

3 牛麗霞;EZH2及RUNX3在子宮腺肌病中的表達及其相關(guān)性[D];延邊大學(xué);2015年

4 李雪;高強度聚焦超聲治療子宮腺肌病121例療效觀察[D];河北醫(yī)科大學(xué);2015年

5 張俊梅;左炔諾孕酮宮內(nèi)緩釋系統(tǒng)治療子宮腺肌病的臨床療效分析[D];河北醫(yī)科大學(xué);2015年

6 邱涵雅;左炔諾孕酮宮內(nèi)緩釋系統(tǒng)對子宮腺肌病療效的臨床觀察[D];河北醫(yī)科大學(xué);2015年

7 馬榮麗;前列腺素F_(2α)受體在子宮腺肌病不同部位的表達及其臨床意義[D];中國人民解放軍醫(yī)學(xué)院;2015年

8 程曉Z，

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