【摘要】：顆粒細胞的增殖與分化對卵泡的發(fā)育至關重要,顆粒細胞功能異常導致卵泡發(fā)育障礙是引發(fā)卵巢內分泌異常的重要原因。越來越多的證據(jù)顯示microRNA參與卵巢顆粒細胞功能的調節(jié)。我們研究發(fā)現(xiàn)miR-181a在小鼠卵巢生長發(fā)育與卵泡生長過程中表達逐漸降低,提示miR-181a在卵泡發(fā)育過程中具有重要調節(jié)作用,但具體分子機制不清。我們通過熒光定量PCR與細胞增殖活力檢測結果證實,隨著外源性miR-181a表達量的增加,小鼠卵巢顆粒細胞(mGCs)的增殖活力明顯降低；腺病毒介導的小鼠顆粒細胞中外源性miR-181a的表達以梯度依賴的方式抑制mGCs中細胞周期蛋白D2 (cyclin D2) mRNA和蛋白的表達以及增殖細胞核抗原(PCNA)蛋白的表達。miR-181a的功能缺失實驗進一步表明mGCs轉染50nM miR-181a inhibitor時,可顯著增加mGCs的增殖活力以及mGCs中cyclin D2的表達。在進一步的機制研究中,經(jīng)熒光素酶報告基因實驗證實miR-181a通過與激活素受體2a (acvr2a) mRNA 3'UTR區(qū)種子序列(TGAATGT)結合抑制熒光素酶報告基因活性；Q-PCR和Western Blot實驗結果進一步證實腺病毒介導的miR-181a高表達可以明顯抑制mGCs中內源性acvr2a mRNA和蛋白的表達；給予niR-181a inhibitor處理后則顯著促進mGCs中acvr2a mRNA和蛋白的表達；在小鼠發(fā)育過程中,其卵巢acvr2a的表達呈現(xiàn)逐漸升高的趨勢,同時伴隨卵泡生長至竇前與竇卵泡期,卵泡中acvr2a mRNA的表達明顯增多,與卵泡中同期miR-181a的表達呈負相關。Q-PCR結果顯示activin A以濃度依賴的方式顯著抑制小鼠顆粒細胞中miR-181a表達；腺病毒介導的顆粒細胞中高表達miR-181a顯著抑制activin A (50ng/mL)促進mGCs的增殖(P0.05)以及cyclin D2的表達。Western Blot實驗進一步證實miR-181a通過降低細胞中內源性acvr2a的表達而抑制activinA誘導的細胞內Smad2蛋白的磷酸化和CYP19Al、P450scc和ESR1基因的表達。臨床樣本調查結果初步顯示,卵巢早衰病人血漿中miR-181a的表達明顯增多。綜上所述,我們首次發(fā)現(xiàn)卵巢早衰病人血漿中miR-181a明顯增高。在研究miR-181a增高可能的病生理意義時,我們通過小鼠試驗進一步發(fā)現(xiàn)miR-181a通過與acvr2a mRNA 3'UTR區(qū)種子序列結合降低內源性acvr2a的表達,并抑制activin A誘導的mGCs的增殖。這為下一步研究卵巢早衰的分子機制提供了新線索。
[Abstract]:The proliferation and differentiation of granulosa cells is very important to the development of follicles. The abnormal function of granulosa cells leads to the disorder of follicular development, which is an important cause of ovarian endocrine abnormalities. More and more evidence suggests that microRNA is involved in the regulation of ovarian granulosa cell function. Our study found that the expression of miR-181a decreased gradually during ovarian growth and follicular growth in mice, suggesting that miR-181a plays an important regulatory role in follicular development, but the specific molecular mechanism is unclear. The results of fluorescence quantitative PCR and cell proliferation activity showed that the proliferation activity of mouse ovarian granulosa cells (mGCs) decreased significantly with the increase of exogenous miR-181a expression. The expression of exogenous miR-181a in mouse granulosa cells mediated by adenovirus inhibited the expression of cell cycle protein D2 (cyclin D2) mRNA and protein and the expression of proliferating cell nuclear antigen (PCNA) protein in mGCs in a gradient-dependent manner. The functional deletion test of miR-181a further showed that when mGCs was transferred into 50nM miR-181a inhibitor, It could significantly increase the proliferation activity of mGCs and the expression of cyclin D2 in mGCs. In further studies, luciferase reporter gene assay confirmed that miR-181a inhibited luciferase reporter gene activity by binding to the seed sequence (TGAATGT) of activin receptor 2A (acvr2a) mRNA 3 'UTR region. The results of Q-PCR and Western Blot further confirmed that the overexpression of miR-181a mediated by adenoviruses could significantly inhibit the expression of endogenous acvr2a mRNA and protein in mGCs, and the expression of acvr2a mRNA and protein in mGCs was significantly promoted after treatment with niR-181a inhibitor. During the development of mice, the expression of acvr2a in ovaries increased gradually, and the expression of acvr2a mRNA in follicles increased significantly with the growth of follicles to the antral and antral follicles. It was negatively correlated with the expression of miR-181a in follicles at the same time. Q-PCR results showed that activin A significantly inhibited the expression of miR-181a in mouse granulosa cells in a concentration-dependent manner. The overexpression of miR-181a in granulosa cells mediated by adenoviruses significantly inhibited the proliferation of mGCs (P 0.05) and the expression of cyclin D2. Western Blot assay further confirmed that miR-181a decreased the intrinsic acvr2a in cells. Inhibition of intracellular Smad2 protein phosphorylation and CYP19Al, induced by activinA Expression of P450scc and ESR1 genes. The results of clinical sample investigation showed that the expression of miR-181a in plasma of patients with premature ovarian failure was significantly increased. To sum up, for the first time, we found a significant increase in plasma miR-181a in patients with premature ovarian failure. In order to study the possible pathophysiological significance of miR-181a, we further found that miR-181a decreased the expression of endogenous acvr2a by binding to the seed sequence of acvr2a mRNA 3 'UTR region, and inhibited the proliferation of mGCs induced by activin A. This provides a new clue for the next step in the study of the molecular mechanism of premature ovarian failure.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R321.1

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