本文關(guān)鍵詞： 子宮內(nèi)膜異位癥 1ncRNA mRNA 子宮內(nèi)膜異位癥 生物信息學(xué)分析 1ncRNA qRT-PCR HOXA9 HOXA10 HOXA11 HOXD10 DNA甲基化　出處：《北京協(xié)和醫(yī)學(xué)院》2014年博士論文　論文類型：學(xué)位論文

【摘要】：雖然目前子宮內(nèi)膜異位癥的病因仍然不明,但一般認(rèn)為它是一種多因素共同導(dǎo)致的疾病,而其中遺傳因素,尤其是表觀遺傳學(xué)調(diào)控在其中起到了重要的作用。比如,DNA甲基化水平改變參與了在內(nèi)異癥中有重要作用的HOXA10和HOXA11基因在在位內(nèi)膜中的表達(dá)下調(diào)。HOXA9和HOXD10基因則被發(fā)現(xiàn)與HOXA10和HOXA11在功能和表達(dá)模式上高度一致。而在本課題組的前期研究中,我們發(fā)現(xiàn)外周血microRNA有作為內(nèi)異癥生物學(xué)標(biāo)記物的潛能。 長鏈非編碼RNA(long non-coding RNA,lncRNA)作為轉(zhuǎn)錄組的重要組成部分,在人類疾病、發(fā)育、干細(xì)胞等領(lǐng)域發(fā)揮了重要的調(diào)控作用。而對1ncRNA在內(nèi)異癥中表達(dá)情況的研究尚屬空白。因此,在本課題中,我們利用微陣列基因芯片技術(shù),探索1ncRNA在卵巢內(nèi)異癥組織和在位內(nèi)膜組織中的表達(dá)差異,建立1ncRNA的表達(dá)譜。在此基礎(chǔ)上,使用生物信息學(xué)方法預(yù)測1ncRNA的潛在功能。 另外,在本研究中我們還探索了DNA甲基化水平異常是否參與了HOXA10,HOXA11, HOXA9和HOXD10基因在內(nèi)異癥組織中相對于在位內(nèi)膜的表達(dá)下調(diào)。 研究目的：在全基因組范圍內(nèi)比較卵巢子宮內(nèi)膜異位癥患者在位及異位內(nèi)膜組織中l(wèi)ncRNA的表達(dá)水平,建立lncRNA的表達(dá)譜。 研究方法：使用高通量的微陣列基因芯片,對4對卵巢內(nèi)異癥患者的在位及異位內(nèi)膜組織同時檢測全基因組范圍內(nèi)lncRNA和mRNA的表達(dá)水平,篩查在位及異位內(nèi)膜組織中差異表達(dá)的lncRNA和mRNA,獲得內(nèi)異癥中l(wèi)ncRNA的表達(dá)譜。 研究結(jié)果：相對于在位內(nèi)膜,卵巢內(nèi)異癥組織有4,088個mRNA轉(zhuǎn)錄本和948個lncRNA轉(zhuǎn)錄本出現(xiàn)了差異表達(dá)。 研究結(jié)論：相對于在位內(nèi)膜組織,mRNA和lncRNA在卵巢內(nèi)異癥組織中表現(xiàn)出了系統(tǒng)性表達(dá)差異。 實驗二部分差異表達(dá)的lncRNA的功能預(yù)測及PCR驗證 研究目的：進(jìn)一步探索1ncRNA在子宮內(nèi)膜異位癥中可能發(fā)揮的功能,同時驗證實驗一基因芯片結(jié)果的可信性。 研究方法：利用已有的mRNA的功能注釋,預(yù)測差異表達(dá)的lncRNA的功能。在實驗一基礎(chǔ)上,擴(kuò)大檢測樣本量,在21對卵巢內(nèi)異癥患者的在位及異位內(nèi)膜組織中,對2組共10個實驗一基因芯片結(jié)果中差異表達(dá)的lncRNA進(jìn)行實時定量聚合酶鏈?zhǔn)椒磻?yīng)(quantitative real-time polymerase chain reaction,qRT-PCR)檢測,以對基因芯片結(jié)果進(jìn)行驗證。 研究結(jié)果：差異表達(dá)的1ncRNA可能參與到了多種生物學(xué)通路中,其中包括數(shù)種已知的內(nèi)異癥發(fā)病機(jī)制中的重要通路。1ncRNA可能通過順式和反式兩種作用方式調(diào)控編碼蛋白的基因,從而發(fā)揮作用。經(jīng)qRT-PCR驗證,lncRNA FN0206、MGC24103、 LOC375295、CHL1-AS2、XL0C_012981、LOC375295和CHL1-AS2表達(dá)上調(diào);LIMS3-LOC440895、LOC389906、HOXA11-AS、KLKP1、LOC100505776和XLOC_012981表達(dá)下調(diào)。 研究結(jié)論：1ncRNA可能通過多種生物學(xué)通路參與到內(nèi)異癥的發(fā)病機(jī)制中。實驗一使用基因芯片檢測1ncRNA表達(dá)的結(jié)果可靠。 實驗三部分卵巢內(nèi)異癥患者在位與異位內(nèi)膜中HOXA9、HOXA10、HOXA11和HOXD10基因甲基化狀態(tài)的研究 研究目的：研究DNA甲基化水平的改變是否可能是導(dǎo)致HOXA9、HOXA10、HOXA11和HOXD10基因在內(nèi)異癥組織中表達(dá)失調(diào)的潛在原因。 研究方法：對HOXA9、HOXA10、HOXA11和HOXD10基因,首先使用qRT-PCR技術(shù)在20對卵巢內(nèi)異癥在位及異位內(nèi)膜樣本中檢測其表達(dá)水平。接下來,使用甲基化芯片技術(shù),在5對樣本中檢測它們啟動子區(qū)的甲基化水平。 研究結(jié)果：HOXA9、HOXA10、HOXA11和HOXD10基因在異位內(nèi)膜組織中的表達(dá)下調(diào)在qRT—PCR實驗中得到了驗證,HOXA10基因CpG島1在異位內(nèi)膜中出現(xiàn)了低甲基化,其他基因未發(fā)現(xiàn)啟動子區(qū)甲基化水平的明顯變化。 研究結(jié)論：HOXA9、HOXA10、HOXA11和HOXD10基因啟動子區(qū)的甲基化水平變化可能不是導(dǎo)致卵巢內(nèi)異癥組織與在位內(nèi)膜組織間表達(dá)水平差異的主要原因。
[Abstract]:Although the cause of endometriosis remains unknown, but it is generally considered a disease caused by multiple factors, including genetic factors, especially the epigenetic regulation has important effect in them. For example, the level of DNA methylation changes in the disease, abnormal expression of HOXA10 gene and HOXA11 gene an important role in the endometrial down-regulation of.HOXA9 and HOXD10 genes were found with HOXA10 and HOXA11 in the expression patterns and function is highly consistent. In ourprevious study, we found that the peripheral blood microRNA of endometriosis as biological markers of potential.
Long chain encoding RNA (long non-coding non RNA, lncRNA) as an important part of the transcriptome development in human disease, and play an important role in the regulation of stem cells and other fields. And the study of 1ncRNA expression in endometriosis is still blank. Therefore, in this study, we used microarray gene chip technology, to explore the differences of expression of 1ncRNA in ovarian endometriosis tissue and eutopic endometrial tissues, expression of 1ncRNA spectrum. On this basis, the potential function of the use of bioinformatics prediction methodology of 1ncRNA.
In addition, in this study, we also explored whether the abnormal DNA methylation level was involved in the downregulation of HOXA10, HOXA11, HOXA9 and HOXD10 genes in endometriosis tissues compared with the eutopic endometrium.
Research purposes: To compare the expression level of lncRNA in eutopic and ectopic endometrium of patients with ovarian endometriosis in the whole genome, and to establish lncRNA expression profile.
Methods: using high throughput microarray gene chip, and to detect the expression of genome wide lncRNA and mRNA of 4 patients with ovarian endometriosis eutopic and ectopic endometrial tissue, differential expression screening of eutopic and ectopic endometrium tissues of lncRNA and mRNA, the expression of lncRNA in different gain spectrum of the disease.
Results: compared with the eutopic endometrium, 4088 mRNA transcripts and 948 lncRNA transcripts showed differential expression in the ovarian endometrium.
Conclusion: compared with the eutopic endometrium, mRNA and lncRNA showed systematic expression differences in the ovarian endometrium.
The functional prediction and PCR verification of the differential expression of lncRNA in the two part of the experiment
Objective: to further explore the possible function of 1ncRNA in endometriosis, and to verify the credibility of the results of a gene chip.
Research methods: using the functional annotation of existing mRNA, predicted the differentially expressed lncRNA functions. In the experimental basis, enlarge the sample detection, in 21 of patients with ovarian endometriosis eutopic and ectopic endometrial tissue, differential expression of 2 groups of a total of 10 experimental results of gene chip lncRNA real-time quantitative polymerase chain reaction (Quantitative real-time polymerase chain reaction, qRT-PCR) to detect, to validate the results of gene chip.
Results: expression of 1ncRNA may be involved in a variety of biological pathways, including.1ncRNA pathway of endometriosis pathogenesis of a number of known in May by the cis - and trans gene two protein encoding regulation mode of action, so as to play a role. Verified by qRT-PCR, lncRNA FN0206, MGC24103, LOC375295, CHL1-AS2, XL0C_012981, upregulation of LOC375295 and CHL1-AS2; LIMS3-LOC440895, LOC389906, HOXA11-AS, KLKP1, LOC100505776 and XLOC_012981 expression.
Conclusion: 1ncRNA may be involved in the pathogenesis of endometriosis through various biological pathways. In Experiment 1, the detection of 1ncRNA expression by gene chip is reliable.
Study on the methylation status of HOXA9, HOXA10, HOXA11 and HOXD10 genes in the three part of experimental ovarian endometriosis patients
Objective: To study whether the change of DNA methylation level may be the underlying cause of HOXA9, HOXA10, HOXA11 and HOXD10 gene expression imbalance in endometriosis.
Methods: HOXA9, HOXA10, HOXA11 and HOXD10 genes, the first use of qRT-PCR technology in 20 to detect the expression of ovarian endometriosis eutopic and ectopic endometrial samples. Then, the use of chip technology in the detection of methylation, 5 methylation level of promoter region of their sample.
Results: the down regulated expression of HOXA9, HOXA10, HOXA11 and HOXD10 genes in ectopic endometrium was verified in the qRT PCR experiment. HOXA10 gene CpG island 1 showed hypomethylation in ectopic endometrium, and no significant change in promoter methylation level was found in other genes.
Conclusion: the methylation level of promoter region of HOXA9, HOXA10, HOXA11 and HOXD10 may not be the main reason for the difference in expression level between ovarian endometriosis and eutopic endometrium.

【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】：2014
【分類號】：R711.71

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