【摘要】：第一部分MicroRNA-22-3p在原發(fā)性卵巢功能不全患者血漿中顯著下調(diào)及意義背景：長度為21-24核苷酸的非編碼RNA被稱為microRNA (miRNA),廣泛存在于人體各種組織和體液中,在轉(zhuǎn)錄后水平調(diào)控基因表達。大量研究證實miRNAs不僅可以作為診斷多種癌癥、糖尿病等的有效標志物,在疾病發(fā)病過程中發(fā)揮重要作用；還可以影響生殖細胞增殖和凋亡,調(diào)控排卵及黃素化部分關(guān)鍵過程,參與卵巢功能的多個方面。目前關(guān)于niRNAs在POI疾病的研究較少,尚不能全面闡明niRNAs在POI患者中的變化及作用。目的：本研究旨在通過μParafloTM MicroRNA芯片技術(shù)篩選POI患者血漿中差異表達的miRNAs,并在較大樣本人群中驗證明確miRNAs在POI患者血漿中的表達譜及其臨床作用。方法：募集2012年9月至2013年12月就診于山東大學附屬生殖醫(yī)院的中國漢族POI患者140例、因男方因素或輸卵管因素就診的內(nèi)分泌正常對照女性140例,留取新鮮血漿。首先通過μParafloTM MicroRNA芯片技術(shù)在10例POI患者和10例對照女性血漿中篩選差異表達的miRNAs,選擇差異顯著的miRNAs在260例獨立樣本中進行驗證。結(jié)果：芯片篩選出差異表達miRNAs共51個,其中22個在POI患者血漿中上調(diào),29個下調(diào)。選擇表達豐度較高的9個差異表達miRNAs (let-7b-5p、let-7c、 miR-15b-5p、miR-22-3p、miR-23a-3p、miR-23b-3p、miR-24-3p、miR-151a-5p和miR-151b)在100例獨立樣本中驗證,發(fā)現(xiàn)niR-22-3p在POI患者血漿中表達量下降,其余8個miRNAs在兩組人群表達無顯著差異。隨后我們在160例樣本中驗證miR-22-3p,結(jié)果顯示miR-22-3p在POI患者血漿中表達顯著降低(差異倍數(shù)：0.74, P0.05); ROC (receiver operating characteristic)分析提示]miR-22-3p區(qū)分POI患者與對照人群的曲線下面積(Area Under Curve, AUC)為0.668；95%confidence interval (CI):0.602-0.733;截斷值(cut off value)為0.607,此截斷值區(qū)分POI患者的敏感性和特異性分別是75.4%和54.6%�；貧w分析表明miR-22-3p是POI保護性因素(OR值：0.766,95%CI：0.643-0.912),與血清FSH水平負相關(guān)(R2=0.687,P0.05)。結(jié)論：本研究在POI患者血漿中篩選差異表達的miRNAs,發(fā)現(xiàn)miR-22-3p在POI患者表達量顯著降低,與血清FSH水平負相關(guān)。提示miR-22-3p可能反映卵巢儲備功能,是POI病理過程的一個結(jié)果。第二部分MicroRNA-379-5p調(diào)控PARP1、XRCC6在生化異常期原發(fā)性卵巢功能不全中的作用和機制研究背景：原發(fā)性卵巢功能不全病因高度異質(zhì),卵子發(fā)生障礙和卵泡耗竭加速是目前POI兩種主要的致病機制假說。顆粒細胞是包繞卵母細胞的體細胞,與卵母細胞之間存在復(fù)雜的調(diào)控網(wǎng)絡(luò),大量研究指出顆粒細胞增殖、分化、侵襲等功能異常是卵巢卵泡耗竭的原因之一,而miRNAs可能在其中發(fā)揮重要作用。已有動物實驗和體外實驗表明miRNAs可以影響顆粒細胞增殖、凋亡及激素合成功能,但尚無POI患者顆粒細胞中miRNAs表達譜研究及功能研究。目的：本課題通過niRCURY?芯片技術(shù)篩選生化異常期POI患者顆粒細胞中差異表達的miRNAs;結(jié)合表達譜芯片結(jié)果,針對差異表達的miRNAs及其可能的靶基因進行功能實驗,研究其對顆粒細胞生物學功能的影響和作用機制,從表觀遺傳角度探討非編碼RNA參與POI發(fā)生發(fā)展過程的分子機理。方法：募集2013年9月至2014年12月就診于山東大學附屬生殖醫(yī)院的中國漢族生化異常階段POI患者50例、因男方因素或輸卵管因素就診的內(nèi)分泌正常對照女性50例,留取顆粒細胞。通過miRCURY?芯片技術(shù)篩選差異表達的miRNAs;與表達譜芯片聯(lián)合分析選擇差異最顯著的miR-379-5p和其可能的靶基因PARP1、 XRCC6,通過Real-Time PCR在獨立樣本顆粒細胞中驗證它們的表達趨勢；在顆粒細胞系中過表達miR-379-5p,分別在未處理狀態(tài)和紫外線(UV)和雙氧水(H202)損傷后通過MTT、CCK8和EdU實驗驗證miR-379-5p對顆粒細胞增殖能力及損傷修復(fù)功能的影響；雙熒光素酶報告系統(tǒng)實驗等驗證niR-379-5p對靶基因PARP1和XRCC6的調(diào)控及機制；沉默顆粒細胞中內(nèi)源性PARP1和XRCC6,重復(fù)上述實驗,明確miR-379-5p降調(diào)PARP1和XRCC6,對顆粒細胞增殖和DNA損傷修復(fù)功能的影響和作用機制。結(jié)果：芯片篩選出差異表達miRNAs共75條。我們對miRNA芯片和表達譜芯片結(jié)果進行聯(lián)合分析,鎖定miR-379-5p和它可能的靶基因PARPU XRCC60在60例獨立樣本中證實miR-379-5p在生化異常期POI患者顆粒細胞中顯著上調(diào),PARPU XRCC6在POI患者顆粒細胞中顯著下調(diào)。過表達顆粒細胞中miR-379-5p可以顯著抑制KGN細胞增殖,其增殖抑制作用呈時間依賴性；在UV/H2O2損傷后,過表達niR-379-5p組KGN細胞增殖抑制作用尤其顯著。Luciferase和western blot實驗證實miR-379-5p通過與PARPUXRCC6的3'UTR區(qū)域結(jié)合,降調(diào)PARPU XRCC6基因。特異性沉默內(nèi)源性PARPU XRCC6基因?qū)GN細胞生物學行為可以產(chǎn)生與過表達miR-379-5p類似的作用,包括抑制KGN細胞增殖、UV/H2O2損傷后較低的細胞存活率和顯著的增殖抑制作用等。結(jié)論：本研究首次發(fā)現(xiàn)POI患者顆粒細胞中顯著上調(diào)的miR-379-5p可能通過降調(diào)PARPU XRCC6影響顆粒細胞增殖及損傷修復(fù)功能,參與POI疾病進程。第三部分長鏈非編碼RNA在生化異常期原發(fā)性卵巢功能不全中的初步研究背景：長鏈非編碼RNA (long noncoding RNA, IncRNA)是由RNA聚合酶Ⅱ轉(zhuǎn)錄形成、長度超過200nt的RNA分子,具有保守的剪接點和內(nèi)含子,在組織和細胞中有特異的表達模式。LncRNAs可以參與染色體重塑、基因轉(zhuǎn)錄的激活和干擾、以及核內(nèi)運輸?shù)戎匾{(diào)控過程,通過表觀遺傳沉默、轉(zhuǎn)錄和轉(zhuǎn)錄后水平調(diào)節(jié)等方式對基因表達進行調(diào)控,近年來,逐漸成為生物醫(yī)學的研究熱點。已有研究證實lncRNAs在癌癥和神經(jīng)系統(tǒng)疾病等人類重大疾病過程中發(fā)揮了重要的調(diào)控作用,但目前已經(jīng)明確功能的lncRNAs僅有數(shù)十條,而在生殖系統(tǒng)疾病領(lǐng)域,尚無相關(guān)研究報道。目的：本研究通過芯片技術(shù)篩選并驗證生化異常階段POI患者顆粒細胞中差異表達的lncRNAs,初步探討lncRNAs在POI患者顆粒細胞中的表達情況。方法：募集2013年9月至2014年12月就診的中國漢族生化異常階段POI患者50例、因男方因素或輸卵管因素就診的內(nèi)分泌正常對照女性50例,留取顆粒細胞。通過LncRNA芯片技術(shù)在10例POI患者與10例對照女性顆粒細胞中篩選差異表達的lncRNAs和mRNAs;根據(jù)表達譜芯片Gene ontology和KEGG pathway分析結(jié)果,對差異表達的lncRNAs和mRNAs進行共表達網(wǎng)絡(luò)分析(co-expression networks),按照相對表達豐度、變化倍數(shù)及序列信息,選擇處于核心位置、與重要且差異表達的基因存在相關(guān)關(guān)系的14條lncRNAs在60例獨立樣本中進行驗證。結(jié)果：芯片結(jié)果發(fā)現(xiàn)差異表達lncRNAs序列677條。Real-Time PCR實驗證實其中：TCONS 00010009、TCONS 00014547、NR 040662.1、ENST00000520306、ENST00000420313、ENST00000526225在POI患者顆粒細胞中顯著下調(diào)。生物信息學分析表明它們?nèi)繉儆趌incRNAs,其周圍均分布有許多功能重要的編碼基因,尤其DNA損傷修復(fù)基因XRCC4和MSH5分別位于TCONS_00010009和NR 040662附近不足300kb處,提示我們這些差異表達的lincRNAs可能通過調(diào)控周圍的共表達基因參與POI疾病。結(jié)論：本研究首次通過Arraystar Human IncRNA芯片對顆粒細胞OncRNAs特征進行初步研究。生化異常階段POI患者顆粒細胞中存在較多差異表達的LncRNA,可能與疾病相關(guān)。
[Abstract]:The first part is the significant down-regulation and significance of MicroRNA-22-3p in the plasma of patients with primary ovarian insufficiency: the non coded RNA with a length of 21-24 nucleotides is called microRNA (miRNA), which widely exists in various tissues and body fluids of the human body and regulates gene expression at post transcriptional level. A large number of studies have confirmed that miRNAs can not only be used as a diagnostic multiple. The effective markers of cancer and diabetes, such as diabetes, play an important role in the pathogenesis of disease; it can also affect the proliferation and apoptosis of germ cells, regulate the key processes of ovulation and yellowing, and participate in many aspects of ovarian function. At present, there are few studies on niRNAs in the disease of POI, and there is no comprehensive explanation of niRNAs in POI patients. Objective: the purpose of this study was to screen the differential expression of miRNAs in the plasma of POI patients by micron ParafloTM MicroRNA chip technology, and to verify the expression and clinical effect of miRNAs in the plasma of POI patients in a large sample population. Methods: to collect the Reproductive Medicine Affiliated to the Shandong University from September 2012 to December 2013. 140 cases of POI patients in Chinese Han nationality, 140 cases of normal control women with endocrine and fallopian tube factors, 140 cases of fresh plasma were left. The differential expression of miRNAs was screened in 10 cases of POI patients and 10 cases of control female plasma by micron MicroRNA chip technique. The difference of significant miRNAs was selected in 260 independent samples. Results: the chip screened a total of 51 differentially expressed miRNAs, of which 22 were up-regulated and 29 down-regulated in the plasma of POI patients. 9 differential expressions of miRNAs (let-7b-5p, Let-7c, miR-15b-5p, miR-22-3p, miR-23a-3p, miR-23b-3p, miR-24-3p, miR-151a-5p and miR-151b) were verified in 100 independent samples. The expression of R-22-3p in the plasma of POI patients decreased and the other 8 miRNAs expressed no significant difference in the two groups. Then we tested the miR-22-3p in 160 samples. The results showed that the expression of miR-22-3p in the plasma of POI patients was significantly reduced (the difference Multiplier: 0.74, P0.05); ROC (receiver operating characteristic) analysis suggested]miR-22-3p area The area under the curve (Area Under Curve, AUC) in the POI patients and the control group was 0.668, 95%confidence interval (CI): 0.602-0.733, and the truncated value (cut off value) was 0.607. 643-0.912) and negative correlation with serum FSH level (R2=0.687, P0.05). Conclusion: This study screened the differential expression of miRNAs in the plasma of POI patients. It was found that the expression of miR-22-3p in POI patients was significantly lower and negatively correlated with the level of serum FSH. It suggested that miR-22-3p may reflect the ovarian reserve power, which is a result of POI pathological process. Second part MicroRNA. -379-5p regulates the role and mechanism of PARP1 and XRCC6 in primary ovarian dysfunction during biochemical abnormal stages: the high heterogeneity of the etiology of primary ovarian dysfunction, the disorder of oogenesis and the acceleration of follicle exhaustion are the two main pathogenesis hypothesis of POI. Granulosa cells are somatic cells wrapped around oocytes and oocytes. There is a complex regulatory network. A large number of studies have pointed out that the proliferation, differentiation and invasion of granulosa cells are one of the reasons for ovarian follicle depletion, and miRNAs may play an important role in it. Animal experiments and in vitro experiments have shown that miRNAs can affect granulosa cell proliferation, apoptosis and hormone synthesis, but there is no POI patient yet. Study and function study of miRNAs expression in granulosa cells. Objective: to screen the differential expression of miRNAs in granulosa cells of patients with POI in biochemical abnormal phase by niRCURY chip technology, and to study the granulosa cell biology by combining the results of the expression spectrum chip to the differentially expressed miRNAs and its possible target genes. The effect and mechanism of function, from the epigenetic point of view, to explore the molecular mechanism of the non coded RNA participating in the development of POI. Methods: 50 cases of POI patients were collected from September 2013 to December 2014 in the affiliated reproductive Hospital of Shandong University. 50 women compared with 50 women, retained granular cells. The differentially expressed miRNAs was screened by miRCURY chip technology; the most significant difference was selected with the expression spectrum chip to select the most significant miR-379-5p and its possible target gene PARP1, XRCC6. The expression trend was verified by Real-Time PCR in the independent sample granulosa cells, and the table in the granular cell line was overexpressed. The effects of miR-379-5p on the proliferation and repair function of granulosa cells were verified by MTT, CCK8 and EdU experiments after the untreated state and ultraviolet (UV) and hydrogen peroxide (H202) damage, respectively. The regulation and mechanism of niR-379-5p on the target gene PARP1 and XRCC6 were verified by the double luciferase reporter system experiment, and the silencing of granular cells was verified by the double luciferase reporter system experiment. Endogenous PARP1 and XRCC6, repeat the above experiments to determine the effect and mechanism of miR-379-5p downfall PARP1 and XRCC6 on the proliferation of granulosa cells and the function of DNA damage repair. Results: the chip screened a total of 75 differentially expressed miRNAs strips. We combined the results of miRNA chip and expression spectrum chip, locked miR-379-5p and its possible target. In 60 independent samples, gene PARPU XRCC60 showed that miR-379-5p was significantly up-regulated in granulosa cells of POI patients with abnormal biochemical phase, and PARPU XRCC6 was significantly down-regulated in granulosa cells of POI patients. MiR-379-5p could significantly inhibit the proliferation of KGN cells in the overexpressed granulosa cells, and its proliferation inhibition was time dependent; after UV/H2O2 damage, a table was overexpressed. The proliferation inhibition effect of KGN cells in the niR-379-5p group was particularly significant.Luciferase and Western blot experiments confirmed that miR-379-5p could reduce the PARPU XRCC6 gene by binding to the 3'UTR region of PARPUXRCC6. N cell proliferation, lower cell survival rate and significant proliferation inhibition after UV/H2O2 injury. Conclusion: This study first found that the significant up regulation of miR-379-5p in granulosa cells of POI patients may be involved in the function of granulosa cell proliferation and injury repair by lowering the modulation of PARPU XRCC6 in the POI disease process. The third part of the long chain non coded RNA is in the process. Primary research background in primary ovarian dysfunction in biochemical abnormal phase: long chain non coded RNA (long noncoding RNA, IncRNA) is transcribed by RNA polymerase II, with a RNA molecule in length exceeding 200nt, with a conservative splicing point and intron, and a specific expression pattern in tissues and cells that.LncRNAs can be involved in chromosome remodeling. The activation and interference of gene transcription, as well as important regulatory processes such as intra nuclear transport, regulate gene expression through epigenetic silence, transcriptional and post transcriptional regulation. In recent years, it has gradually become a research hotspot in biomedicine. LncRNAs has been proved to be in the process of human major diseases such as cancer and nervous system diseases. It has played an important role in regulation and control, but there are only dozens of lncRNAs clearly defined functions, and there are no related reports in the field of reproductive system disease. Objective: This study screened and verified the differential expression of lncRNAs in granulosa cells of patients with POI in biochemical abnormal stage by chip technology, and preliminarily explored the granulosa cells of lncRNAs in POI patients. Methods: 50 cases of POI patients were collected from September 2013 to December 2014 in the Chinese Han nationality biochemical abnormal stage. 50 cases of endocrine normal control women were treated with male and fallopian factors and 50 cases of granulosa cells were left. The difference table was screened in 10 cases of POI patients and 10 control female granulosa cells through the technique of POI chip. LncRNAs and mRNAs, according to the analysis results of Gene ontology and KEGG pathway on the expression spectrum chip, the co expression network analysis (co-expression networks) of the differentially expressed lncRNAs and mRNAs, according to the relative expression abundance, the multiplier and sequence information, is selected at the core position and is related to the important and differentially expressed genes. 14 lncRNAs were verified in 60 independent samples. Results: the results showed that 677.Real-Time PCR experiments of differential expression lncRNAs sequence showed that TCONS 00010009, TCONS 00014547, NR 40662.1, ENST00000520306, ENST00000420313, ENST00000526225 were significantly downregulated in POI patient granulosa cells. Bioinformatics analysis table All of them belong to lincRNAs, and there are many important coding genes around them. Especially, the DNA damage repair gene XRCC4 and MSH5 are located at the deficiency of 300KB near TCONS_00010009 and NR 040662, suggesting that these differentially expressed lincRNAs may participate in the POI disease by regulating the surrounding co expression genes. The initial study on the OncRNAs characteristics of granulosa cells by Arraystar Human IncRNA chip is the first time. There are many differentially expressed LncRNA in the granulosa cells of POI patients at the biochemical abnormal stage, which may be related to the disease.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R711.75

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4 翁靜;諸定壽;;小鼠卵周圍顆粒細胞排卵后在輸卵管內(nèi)死亡過程的研究[A];第九次全國生殖生物學學術(shù)研討會論文摘要集[C];2003年

5 沈浣;;顆粒細胞與卵母細胞發(fā)育[A];中華醫(yī)學會第六次全國生殖醫(yī)學學術(shù)會議�？痆C];2012年

6 王春生;李晶;張滕子;寧方勇;樸善花;安鐵洙;;擬蜘蛛絲蛋白基因在小鼠顆粒細胞中的整合及其鑒定[A];中國畜牧獸醫(yī)學會動物解剖學及組織胚胎學分會第十六次學術(shù)研討會論文集[C];2010年

7 甄t熑

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