【摘要】：研究背景及目的子宮腺肌病是女性常見(jiàn)疾病,主要臨床表現(xiàn)為不同程度的繼發(fā)性進(jìn)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(jī)制仍不清楚。目前關(guān)于其發(fā)病機(jī)制存在多種學(xué)說(shuō),最廣為接受的學(xué)說(shuō)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(gè)方面。但是編碼基因相關(guān)的研究較為零散,通過(guò)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(jī)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(dá)調(diào)控中發(fā)揮重要的作用,并參與多種重要信號(hào)通路的調(diào)節(jié)。其異常表達(dá)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(wú)研究報(bào)道。本研究的目的為：1. 構(gòu)建子宮腺肌病在位內(nèi)膜與對(duì)照內(nèi)膜lncRNAs和nRNAs的差異表達(dá)譜；2. 對(duì)lncRNA和mRNA的功能進(jìn)行生物學(xué)信息分析,以探討其可能的調(diào)控機(jī)制并得出潛在的核心調(diào)節(jié)基因；3. 對(duì)部分潛在核心調(diào)節(jié)基因的表達(dá)水平進(jìn)行檢測(cè),為后續(xù)功能實(shí)驗(yàn)提供研究基礎(chǔ)。研究方法1. 利用基因芯片技術(shù)檢測(cè)子宮腺肌病在位內(nèi)膜和對(duì)照內(nèi)膜組織中l(wèi)ncRNAs和mRNAs的表達(dá),構(gòu)建lncRNAs和mRNAs差異表達(dá)譜(其中兩組的樣本量分別為4例)。2. 利用實(shí)時(shí)熒光定量PCR技術(shù)驗(yàn)證芯片結(jié)果中部分差異表達(dá)IncRNAs(共6個(gè)),以進(jìn)一步驗(yàn)證基因芯片實(shí)驗(yàn)結(jié)果。3. 利用生物信息學(xué)手段對(duì)差異表達(dá)的lncRNAs和mRNAs進(jìn)行分析。3.1 GO分析：對(duì)芯片結(jié)果中差異mRNAs進(jìn)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(jìn)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(dá)網(wǎng)絡(luò)：通過(guò)實(shí)驗(yàn)組與對(duì)照組比較得到的差異mRNAs、差異lncRNAs在芯片中的實(shí)測(cè)表達(dá)值,分別構(gòu)建實(shí)驗(yàn)組與對(duì)照組的共表達(dá)網(wǎng)絡(luò),通過(guò)比較共表達(dá)網(wǎng)絡(luò)上的差異來(lái)定位兩組樣本中的潛在核心調(diào)節(jié)基因。4. 利用實(shí)時(shí)熒光定量PCR和免疫組化法檢測(cè)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(dá)。研究結(jié)果1. 子宮腺肌病在位內(nèi)膜與對(duì)照內(nèi)膜相比,存在165個(gè)差異表達(dá)lncRNAs和61 2個(gè)差異表達(dá)mRNAs。在差異表達(dá)的lncRNAs中有117個(gè)lncRNAs表達(dá)下調(diào)和48個(gè)lncRNAs表達(dá)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(dá)的mRNAs中,下調(diào)的nRNAs,總數(shù)為414個(gè),上調(diào)的mRNAs總數(shù)198個(gè)。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(gè)lncRNAs進(jìn)行實(shí)時(shí)熒光定量PCR驗(yàn)證,結(jié)果顯示n333955、n337373、n338909這3個(gè)lncRNAs在子宮腺肌病在位內(nèi)膜組織中表達(dá)水平下降,而n341651、n342794、n387706表達(dá)水平升高,差異均具有統(tǒng)計(jì)學(xué)意義,與芯片檢測(cè)的表達(dá)趨勢(shì)相符。3. 生物學(xué)信息分析結(jié)果：3.1通過(guò)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(xiàng),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(hào)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(xiàng),包括：基因表達(dá)、DNA復(fù)制、細(xì)胞分裂、有絲分裂細(xì)胞周期的G1/S期過(guò)渡、免疫反應(yīng)等。3.2通過(guò)顯著性Pathway分析,得到上調(diào)差異基因參與的顯著性信號(hào)轉(zhuǎn)導(dǎo)通路共40項(xiàng),包括MAPK信號(hào)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(hào)通路等。下調(diào)差異基因參與的顯著性信號(hào)轉(zhuǎn)導(dǎo)通路共39項(xiàng),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(dá)網(wǎng)絡(luò),兩張共表達(dá)網(wǎng)絡(luò)的結(jié)構(gòu)存在明顯差別。根據(jù)基因在兩個(gè)網(wǎng)絡(luò)中共表達(dá)地位的變化情況,篩選出對(duì)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(shí)時(shí)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(dá)明顯高于對(duì)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(dá)明顯低于對(duì)照內(nèi)膜組織,差異均具有統(tǒng)計(jì)學(xué)意義。研究結(jié)論1. 首次通過(guò)芯片檢測(cè)成功構(gòu)建子宮腺肌病在位內(nèi)膜與對(duì)照內(nèi)膜lncRNAs和mRNAs的差異表達(dá)譜。2. 實(shí)時(shí)熒光定量PCR結(jié)果與芯片結(jié)果有很好的一致性,進(jìn)一步驗(yàn)證芯片結(jié)果的可信度。3. 通過(guò)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(guò)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(dá)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(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R711.71

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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

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8 程曉Z，

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