本文選題：多囊卵巢綜合征 + microRNA��； 參考：《吉林大學(xué)》2016年博士論文

【摘要】：多囊卵巢綜合征(polycystic ovary syndrome,PCOS)是一種復(fù)雜的、異質(zhì)性?xún)?nèi)分泌紊亂綜合征,以高雄激素血癥、高胰島素血癥、胰島素抵抗和持續(xù)性無(wú)排卵為主要特征。PCOS患者的臨床表現(xiàn)多樣,其近期和遠(yuǎn)期并發(fā)癥對(duì)女性的身心健康影響較大。據(jù)報(bào)道,PCOS是無(wú)排卵性不孕的最常見(jiàn)原因,另外PCOS患者2型糖尿病、心血管疾病和子宮內(nèi)膜癌的發(fā)病風(fēng)險(xiǎn)與非PCOS患者相比明顯升高。鑒于PCOS對(duì)女性健康的巨大危害,對(duì)其發(fā)病機(jī)制的研究已經(jīng)成為全球婦科內(nèi)分泌專(zhuān)家關(guān)注的焦點(diǎn)。目前認(rèn)為PCOS可能的致病原因包括雄激素生成過(guò)多、下丘腦垂體功能紊亂、胰島素抵抗、細(xì)胞凋亡及卵泡發(fā)育異常等,其中細(xì)胞凋亡和卵泡發(fā)育異常導(dǎo)致PCOS發(fā)病的理論受到越來(lái)越多學(xué)者的關(guān)注,但是具體的發(fā)病機(jī)制尚需要進(jìn)一步挖掘。微小RNA(micro RNA,mi RNA)是一類(lèi)內(nèi)源性單鏈、非編碼的小分子RNA,一般長(zhǎng)度為18-24核苷酸,通過(guò)與其靶基因3’非編碼區(qū)(untranslated region,UTR)結(jié)合,在轉(zhuǎn)錄后水平負(fù)性調(diào)控其靶基因。近年來(lái),人們發(fā)現(xiàn)mi RNA在子宮、輸卵管、卵巢等女性生殖器官中均有表達(dá),并廣泛參與調(diào)控女性卵泡發(fā)育成熟、受精、著床及胚胎發(fā)育等生理過(guò)程,對(duì)維持女性正常生育功能起重要作用。同時(shí)研究還發(fā)現(xiàn),mi RNA的異常表達(dá)可能與很多女性疾病的發(fā)生有關(guān),如PCOS患者卵泡顆粒細(xì)胞中的mi RNA存在異常表達(dá),而其中一部分mi RNAs(如let-7a、let-7i和mi R-92b)的異常表達(dá)可能與卵泡顆粒細(xì)胞凋亡有關(guān)。由此可見(jiàn),mi RNA可能通過(guò)影響顆粒細(xì)胞凋亡,進(jìn)而導(dǎo)致PCOS的發(fā)生。本研究的目的在于揭示對(duì)PCOS發(fā)病起關(guān)鍵作用的mi RNA及其導(dǎo)致疾病發(fā)生的調(diào)控作用機(jī)制。由于PCOS患者顆粒細(xì)胞取材困難,因此本研究構(gòu)建了PCOS大鼠模型,借助mi RNA深度測(cè)序、熒光定量PCR、Western Blot、基因轉(zhuǎn)染和雙熒光素酶報(bào)告基因檢測(cè)等實(shí)驗(yàn)技術(shù),挖掘可能與PCOS疾病發(fā)生相關(guān)的mi RNAs及其下游調(diào)控通路。主要研究?jī)?nèi)容如下:1.PCOS大鼠模型的構(gòu)建我們采用來(lái)曲唑灌胃法建立PCOS大鼠模型。PCOS模型組:每日將來(lái)曲唑按1mg/(kg?d)溶解到1%羧甲基纖維素(CMC)中,連續(xù)23天灌胃處理;對(duì)照組:每日CMC 1mg/(kg?d),連續(xù)23天灌胃處理。結(jié)果:來(lái)曲唑灌胃處理后,大鼠陰道涂片顯示,對(duì)照組大鼠存在規(guī)律的動(dòng)情周期變化,而模型組大鼠的動(dòng)情周期失去規(guī)律性變化,模型組大鼠的陰道涂片可見(jiàn)大量白細(xì)胞,提示大鼠持續(xù)處于動(dòng)情間期。卵巢組織HE染色顯示模型組卵巢結(jié)構(gòu)紊亂,卵巢內(nèi)囊狀擴(kuò)張的卵泡增多、黃體及發(fā)育階段卵泡數(shù)目明顯減少。ELISA檢測(cè)顯示,模型組大鼠血清LH、FSH、T濃度顯著高于對(duì)照組,E2濃度顯著低于對(duì)照組。以上實(shí)驗(yàn)數(shù)據(jù)說(shuō)明我們成功構(gòu)建了PCOS大鼠模型。2.深度測(cè)序及生物信息學(xué)分析提取PCOS模型組與對(duì)照組大鼠卵巢組織的總RNA,經(jīng)深度測(cè)序后發(fā)現(xiàn),顯著差異表達(dá)的mi RNAs共有129個(gè),其中表達(dá)上調(diào)的mi RNAs有49個(gè),表達(dá)下調(diào)的mi RNAs有80個(gè)。選取4個(gè)與細(xì)胞增殖、凋亡相關(guān)的mi RNAs進(jìn)行驗(yàn)證,熒光定量PCR結(jié)果顯示,mi R-34b-5p、mi R-141-3p和mi R-200a-3p在模型組大鼠卵巢組織中呈顯著性下調(diào),而mi R-201-5p在模型組大鼠卵巢組織中呈顯著性上調(diào),進(jìn)一步驗(yàn)證了深度測(cè)序結(jié)果的準(zhǔn)確性。隨后利用生物信息學(xué)軟件對(duì)4個(gè)差異表達(dá)mi RNAs進(jìn)行下游靶基因預(yù)測(cè),共發(fā)現(xiàn)2060個(gè)靶基因。運(yùn)用Gene Ontology(GO)、Pathway analysis等軟件對(duì)這些靶基因的潛在下游調(diào)控通路進(jìn)行富集和篩選,發(fā)現(xiàn)4個(gè)差異表達(dá)mi RNAs的靶基因參與卵母細(xì)胞減數(shù)分裂、MAPK信號(hào)通路、PI3K-Akt信號(hào)通路、Rap1信號(hào)通路及Notch信號(hào)通路、生殖過(guò)程和細(xì)胞凋亡等多條信號(hào)通路。3.選取mi R-141-3p進(jìn)行功能驗(yàn)證mi R-141-3p在PCOS模型組卵巢中的表達(dá)水平呈顯著性下調(diào)。MTT法顯示,過(guò)表達(dá)mi R-141-3p后,細(xì)胞活力明顯增強(qiáng);干擾mi R-141-3p功能后,細(xì)胞活力明顯減弱。流式細(xì)胞術(shù)顯示,過(guò)表達(dá)mi R-141-3p后,細(xì)胞促凋亡的能力明顯減弱;干擾mi R-141-3p功能后,細(xì)胞促凋亡的能力明顯增強(qiáng)。生物信息學(xué)分析軟件預(yù)測(cè)結(jié)果顯示死亡相關(guān)蛋白激酶1(death-associated protein kinase,DAPK1)的3’-UTR區(qū)含有可與mi RNA-141-3p互補(bǔ)結(jié)合的序列,DAPK1可能是mi R-141-3p的靶基因。熒光定量PCR結(jié)果顯示,與對(duì)照組相比,模型組大鼠卵巢中mi R-141-3p顯著性下調(diào),而DAPK1顯著性上調(diào),二者在PCOS大鼠卵巢中的表達(dá)呈明顯負(fù)相關(guān)。使用mi R-141-3p模擬劑和抑制劑轉(zhuǎn)染大鼠卵巢顆粒細(xì)胞后,DAPK1 m RNA和蛋白水平均與mi R-141-3p表達(dá)水平呈負(fù)相關(guān)。雙熒光素酶報(bào)告基因?qū)嶒?yàn)顯示,當(dāng)HEK 293T細(xì)胞轉(zhuǎn)染含Wt-DAPK1 3’-UTR的熒光素酶報(bào)告載體時(shí),mi R-141-3p模擬劑組的熒光素酶活性較對(duì)照組明顯降低;而當(dāng)HEK 293T細(xì)胞轉(zhuǎn)染含Mut-DAPK1 3’-UTR的熒光素酶報(bào)告載體時(shí),mi R-141-3p模擬劑組和對(duì)照組的熒光素酶活性無(wú)顯著性差異。上述結(jié)果表明,mi R-141-3p能與DAPK1基因3’-UTR結(jié)合,并對(duì)DAPK1轉(zhuǎn)錄有負(fù)性調(diào)控作用,證實(shí)DAPK1是mi R-141-3p的靶基因。結(jié)果表明,PCOS大鼠模型卵巢中mi RNA的異常表達(dá)可能是引起PCOS發(fā)病的重要原因。PCOS大鼠模型卵巢中低表達(dá)的mi R-141-3p通過(guò)靶向調(diào)控DAPK1的表達(dá),進(jìn)而在PCOS發(fā)生發(fā)展中發(fā)揮促進(jìn)細(xì)胞凋亡的作用,mi R-141-3p/DAPK1有望成為新的PCOS診斷標(biāo)志物和治療靶點(diǎn)。
[Abstract]:Polycystic ovary syndrome (PCOS) is a complex, heterogeneous endocrine disorder syndrome. The clinical manifestations of the patients with.PCOS, Kaohsiung, hyperinsulinemia, insulin resistance and persistent anovulatory, are diverse, and their close and long-term complications have a greater impact on the physical and mental health of women. It is reported that PCOS is the most common cause of anovulatory infertility. In addition, the risk of type 2 diabetes, cardiovascular disease and endometrial cancer in PCOS patients is significantly higher than that of non PCOS patients. In view of the great harm of PCOS to women's health, the study of its pathogenesis has become the focus of the whole gynecologic endocrinologist. The possible causes of PCOS include excessive androgen formation, hypothalamic pituitary dysfunction, insulin resistance, apoptosis and follicular development. The theory of apoptosis and follicle development caused by abnormal follicle development is concerned by more and more scholars, but the specific pathogenesis needs to be further excavated. Small RNA (micro) RNA, MI RNA) is a class of endogenous single chain, non coded small molecule RNA, with a general length of 18-24 nucleotides, which is negatively regulated by the target gene 3 'non coding region (untranslated region, UTR). In recent years, it has been found that MI RNA is expressed in female reproductive organs such as uterus, fallopian tubes, and ovaries. The extensive participation in regulating the growth and maturation of female follicles, fertilization, implantation and embryo development plays an important role in maintaining the normal reproductive function of women. At the same time, the abnormal expression of MI RNA may be related to the occurrence of many female diseases, such as the abnormal expression of MI RNA in the follicle granulocyte of PCOS patients, and one of them The abnormal expression of MI RNAs (such as let-7a, let-7i and MI R-92b) may be related to the apoptosis of follicle granulosa cells. Thus, MI RNA may affect the occurrence of PCOS by affecting the apoptosis of granulosa cells. The purpose of this study is to reveal the MI RNA and the mechanism of the regulation of the pathogenesis of PCOS. In this study, the PCOS rat model was constructed, and the MI RNA depth sequencing, fluorescence quantitative PCR, Western Blot, gene transfection and double luciferase reporter gene detection were used to excavate mi RNAs and its downstream regulation pathway related to the occurrence of PCOS disease. The main contents are as follows: 1.PCOS rats PCOS rat model.PCOS model group was established by letrozole gavage: PCOS (kg? D) was dissolved into 1% carboxymethyl cellulose (CMC) in the future for 23 days. The control group: CMC 1mg/ (kg? D) daily for 23 days. Results: after letrozole gavage, the rat vagina smear showed the control group big The estrous cycle of the rats was changed, and the estrous cycle of the model group was lost regularly. A large number of leukocytes were found in the vaginal smear of the model group, suggesting that the rats were in the estrus interval. The ovarian tissue HE staining showed that the ovarian structure was disorder, the follicle in the ovary expanded in the ovary, and the follicle in the corpus luteum and the developmental stage. The.ELISA detection showed that the concentration of serum LH, FSH, and T in the model group was significantly higher than that of the control group, and the concentration of E2 was significantly lower than that of the control group. The above experimental data indicated that we successfully constructed the PCOS rat model.2. depth sequencing and bioinformatics analysis to extract the total RNA of the ovarian tissue of the PCOS model group and the control group, and were sequenced by deep sequencing. There were 129 significant differences in the expression of MI RNAs, of which 49 were up - regulated mi RNAs, and 80 down regulated mi RNAs. The MI RNAs, which was associated with cell proliferation and apoptosis, was selected. The fluorescence quantitative PCR results showed that MI R-34b-5p. Mi R-201-5p was significantly up-regulated in the rat ovarian tissue of the model group, which further verified the accuracy of the depth sequencing results. Then, 4 differentially expressed mi RNAs were predicted by bioinformatics software, and 2060 target genes were detected. Gene Ontology (GO), Pathway analysis and other software were used for these target genes. The potential downstream regulation pathways were enriched and screened, and 4 target genes differentially expressed in MI RNAs were involved in meiosis, MAPK signaling pathway, PI3K-Akt signaling pathway, Rap1 signaling pathway and Notch signaling pathway, reproductive process and apoptosis,.3. selected mi R-141-3p to perform functional verification of MI R-141-3p mi R-141-3p in PCOS The expression level in the ovaries of the model group showed a significant downregulation by.MTT method. After overexpression of MI R-141-3p, the cell viability was obviously enhanced. After interfering with the function of MI R-141-3p, the cell viability was markedly weakened. The flow cytometry showed that the ability to promote apoptosis after overexpression of MI R-141-3p was obviously weakened; and the ability to promote apoptosis after MI R-141-3p function was interfered with the function of MI R-141-3p. The results of the bioinformatics analysis software predicted that the 3 '-UTR region of the death related protein kinase 1 (death-associated protein kinase, DAPK1) contains a sequence that can be complementary to MI RNA-141-3p, and DAPK1 may be the target gene of MI R-141-3p. -141-3p significantly downregulated and DAPK1 significantly increased. The expression of two in the ovary of PCOS rats was negatively correlated. DAPK1 m RNA and protein levels were negatively correlated with the MI R-141-3p expression level after transfecting rat ovarian granulosa cells with MI R-141-3p analogue and inhibitor. The double luciferase reporter gene experiment showed that HEK 293T was thin. When cell transfected with Wt-DAPK1 3 '-UTR luciferase reporter carrier, the luciferase activity of MI R-141-3p analogue group was significantly lower than that of the control group, but when HEK 293T cells transfected to the luciferase reporter carrier containing Mut-DAPK1 3' -UTR, there was no significant difference between the luciferase activity of the MI R-141-3p simulation group and the control group. Mi R-141-3p can be combined with DAPK1 gene 3 '-UTR and has a negative regulation on DAPK1 transcription. It is proved that DAPK1 is the target gene of MI R-141-3p. The results show that the abnormal expression of MI RNA in the ovaries of the PCOS rat model may be an important cause of the pathogenesis of PCOS, and the low expression of the rat model ovary is regulated by the target. The expression of PCOS R-141-3p/DAPK1 may play an important role in promoting apoptosis in the development of MI. R-141-3p/DAPK1 is expected to become a new diagnostic marker and therapeutic target for PCOS.

【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R711.75;R-332

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