發(fā)布時間：2018-03-28 17:31

  本文選題：PDCD4　切入點：多囊卵巢綜合征　出處：《山東大學》2016年博士論文

【摘要】：背景多囊卵巢綜合征(Polycystic ovary syndrome,PCOS)是育齡期婦女常見的一種內(nèi)分泌代謝異常性疾病,以慢性無排卵、高雄激素血癥和卵巢多囊樣改變?yōu)橹饕卣鳌３Ｒ姷呐R床表現(xiàn)有不孕、多毛、月經(jīng)紊亂以及肥胖等,其遠期并發(fā)癥包括Ⅱ型糖尿病、心血管疾病等。PCOS婦女肥胖的發(fā)病率為47%,顯著高于普通人群,且肥胖程度與胰島素抵抗(Insulin resistance,IR)呈高度正相關。對肥胖型和非肥胖型PCOS婦女的比較研究發(fā)現(xiàn),肥胖型較非肥胖型PCOS婦女存在更嚴重的代謝紊亂,肥胖加重了IR程度。阻斷肥胖、IR,打破代謝間的惡性循環(huán),對阻斷PCOS進程、改善內(nèi)分泌異常及恢復排卵具有重要意義。育齡期婦女卵泡在其發(fā)育過程中受多種激素的影響,使卵泡經(jīng)歷發(fā)育、成熟、最終排卵。每一月經(jīng)周期只有一個卵泡成熟排卵,其余卵泡則在不同時期閉鎖。大量研究表明卵泡閉鎖與顆粒細胞凋亡有關。在PCOS婦女中卵巢顆粒細胞凋亡明顯增加。PDCD4(programed cell death 4)是近年來新發(fā)現(xiàn)的糖脂代謝相關基因和促凋亡調(diào)控基因,其基因缺失能夠明顯改善胰島素敏感性,抑制肥胖癥的發(fā)生,促進脂質(zhì)代謝,抑制細胞凋亡。PDCD4在PCOS婦女體內(nèi)是否存在著異常表達從而進一步導致其糖脂代謝紊亂、顆粒細胞凋亡增強,將有助于揭示PCOS代謝異常及卵泡發(fā)育障礙的發(fā)生機制,對明確PCOS的發(fā)生發(fā)展有著重要的臨床意義。本研究擬通過測定肥胖型和非肥胖型PCOS婦女以及對照人群外周血PDCD4的表達特點,探討PDCD4與PCOS婦女的體重指數(shù)(Body mass index,BMI)、胰島素抵抗以及脂質(zhì)代謝紊亂的相關性；通過研究PDCD4對PCOS婦女卵巢顆粒細胞凋亡的影響,探討PCOS卵泡發(fā)育障礙的分子機制,為PCOS的促排卵治療提供新的治療思路；進一步觀察應用二甲雙胍改善代謝紊亂后PDCD4基因表達水平的變化,明確PDCD4與PCOS代謝異常的相關性,為闡明PCOS發(fā)生機制提供理論依據(jù),為PCOS的個體化治療提供實驗依據(jù)。目的1.通過比較PDCD4在PCOS婦女以及對照人群中的表達狀態(tài),明確PDCD4在PCOS患者中的表達特點；2.分析PDCD4表達與PCOS婦女BMI、胰島素抵抗以及脂代謝參數(shù)的相關性；應用二甲雙胍改善胰島素敏感性后檢測PDCD4表達水平的變化,闡明PDCD4表達與PCOS內(nèi)分泌代謝紊亂是否存在相關性；3.探討PDCD4表達對PCOS婦女卵巢顆粒細胞凋亡的影響及其作用機制。方法1.病例收集選取在山東大學附屬生殖醫(yī)院就診的PCOS婦女77例,PCOS的診斷依據(jù)中國PCOS診斷標準；另選擇月經(jīng)周期正常、因輸卵管因素或男方因素就診的不孕婦女67例為對照組；所有研究對象排除Cushing綜合征、甲狀腺功能障礙、分泌雄激素腫瘤和Ⅰ型、ⅡI型糖尿病,入組前3個月均未接受過任何藥物或手術治療；根據(jù)BMI分別將PCOS婦女及其對照組分成肥胖亞組(≥25kg/m2)以及非肥胖亞組(25kg/m2)。2.實時熒光定量Real time RT-PCR檢測分離所有病例外周血單個核細胞,提取RNA,利用實時熒光定量Real time RT-PCR檢測PCOS婦女及其對照組PDCD4 mRNA的表達情況。3.卵巢顆粒細胞分離在超聲探頭引導下,經(jīng)陰道穿刺抽吸卵泡取卵,抽吸出的卵泡液在實體顯微鏡下找到卵冠丘復合物,將揀卵后的卵泡液迅速轉(zhuǎn)移至實驗室進行顆粒細胞的洗滌純化。4. Small interfering RNA (SiRNA)轉(zhuǎn)染設計PDCD4-SiRNA序列,利用脂質(zhì)體轉(zhuǎn)染分離純化后的PCOS婦女卵巢顆粒細胞；同時應用與PDCD4序列非同源性序列作為PDCD4-SiRNA陰性對照。5.流式細胞術檢測PCOS婦女及對照組卵巢顆粒細胞分離純化后,立即用Annexin V-FITC和PI(propidium iodide)凋亡檢測試劑盒染色,經(jīng)流式細胞儀檢測分析卵巢顆粒細胞凋亡率。6. TUNEL檢測PCOS婦女卵巢顆粒細胞分離純化后進行PDCD4-SiRNA轉(zhuǎn)染,利用TUNEL凋亡檢測試劑盒染色,經(jīng)激光共聚焦顯微鏡觀察PDCD4-SiRNA轉(zhuǎn)染前后卵巢顆粒細胞凋亡變化情況。7. Western blot檢測PCOS婦女卵巢顆粒細胞分離純化后進行PDCD4-SiRNA轉(zhuǎn)染,提取細胞蛋白測定蛋白濃度后依次行SDS-PAGE電泳,轉(zhuǎn)膜,抗體結(jié)合,暗室顯影,觀察PDCD4-SiRNA轉(zhuǎn)染前后卵巢顆粒細胞凋亡信號通路分子Bax和Bcl-2的表達變化情況。8.統(tǒng)計學分析所有數(shù)據(jù)均使用SPSS統(tǒng)計學軟件包分析,計量資料以均數(shù)±標準差表示,計數(shù)資料以數(shù)值和百分比表示；統(tǒng)計分析采用單因素方差、t檢驗、Spearman相關以及多因素回歸分析；所有假設檢驗均進行雙側(cè)檢驗,P0.05被認為差異有統(tǒng)計學意義。結(jié)果一、PDCD4在PCOS婦女與對照人群中的不同表達狀態(tài)1.PCOS患者與對照組臨床特點的比較PCOS患者雌二醇(E2)、總睪酮(TT)、黃體生成素(LH)、空腹胰島素(Insulin 0)、口服葡萄糖兩小時后胰島素(Insulin 120)以及穩(wěn)態(tài)模型評估胰島素抵抗指數(shù)(HOMA-IR)明顯高于對照組,結(jié)果具有統(tǒng)計學差異(P0.05)；PCOS婦女卵泡刺激素(FSH)明顯低于對照組,結(jié)果具有統(tǒng)計學差異(P0.05)；兩組間年齡、BMI、甲狀腺刺激素(TSH)、催乳素(PRL)、空腹血糖(Glucose0)、口服葡萄糖兩小時后血糖(Glucose 120)、胰島p細胞功能指數(shù)(HOMA-p),總膽固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)沒有統(tǒng)計學差異。2. PCOS婦女與對照組肥胖和非肥胖亞組臨床特點的比較PCOS婦女肥胖亞組E2、TT, LH、Insulin 0、Insulin 120、Glucose 120以及HOMA-IR明顯高于對照肥胖亞組,具有統(tǒng)計學差異(P0.05);FSH明顯低于對照組,結(jié)果具有統(tǒng)計學差異(P0.05)；兩亞組間年齡、BMI、TSH、 PRL、Glucose 0、HOMA-β、TC、TG、LDL和HDL沒有統(tǒng)計學差異。PCOS婦女非肥胖亞組TSH、E2、TT以及LH明顯高于對照非肥胖亞組,具有統(tǒng)計學差異(P0.05);FSH明顯低于對照組,結(jié)果具有統(tǒng)計學差異(P0.05)；兩亞組間年齡、BMI、PRL、Insulin 0、Insulin 120、Glucose 0、Glucose 120、 HOMA-IR、HOMA-β、TC、TG、LDL和HDL沒有統(tǒng)計學差異。3. PCOS婦女與對照組間及其肥胖和非肥胖亞組間PDCD4表達的比較通過對PCOS婦女與對照組病例外周血單個核細胞(PBMCs)中PDCD4 mRNA實時熒光定量PCR檢測分析發(fā)現(xiàn),PDCD4在PCOS婦女中的表達高于對照組,結(jié)果具有統(tǒng)計學差異(P0.05);PCOS婦女肥胖亞組PDCD4表達明顯高于對照肥胖亞組,具有顯著的統(tǒng)計學差異(P0.01);PCOS婦女與對照非肥胖亞組間PDCD4表達沒有統(tǒng)計學差異。二、PDCD4表達與PCOS婦女及其對照組糖脂代謝的相關性分析及其臨床意義1.PDCD4表達與體重指數(shù)(BMI)的相關性PCOS婦女中PDCD4表達與BMI具有顯著的相關性(r=0.52,P0.0001),對于對照組PDCD4表達與BMI沒有相關性。2. PDCD4表達與胰島素抵抗的相關性PCOS婦女中PDCD4表達與Insulin 0、Insulin 120、Glucose 120、HOMA-IR和HOMA-β具有顯著的相關性(r=0.34,0.31,0.29,0.36,0.25；P0.01或者0.05)；對于對照組PDCD4表達與胰島素抵抗指標沒有任何相關性。針對IR的PCOS患者給予口服二甲雙胍三個月治療,通過對其PBMCs中PDCD4 mRNA實時熒光定量PCR檢測發(fā)現(xiàn),PDCD4在二甲雙胍治療后其表達明顯降低,結(jié)果具有統(tǒng)計學差異(P0.05)。3. PDCD4表達與脂代謝紊亂的相關性PCOS婦女中PDCD4表達與TG具有顯著的正相關性(r=0.39；P0.001),PDCD4表達與HDL具有顯著的負相關性(r=-0.41；P0.001),對于對照組PDCD4表達與脂代謝指標沒有任何相關性。4. PDCD4表達與PCOS婦女發(fā)病風險的關聯(lián)性分析通過多因素回歸分析發(fā)現(xiàn),校正E2和Insulin 120后,PDCD4表達對于PCOS婦女發(fā)病是獨立的風險因素(相對危險系數(shù),1.318；95%可信區(qū)間,1.021-1.703,P0.05),表明PDCD4的高表達加重了PCOS的發(fā)生和發(fā)展,是反映PCOS婦女發(fā)病的重要危險因素。三、PDCD4表達對PCOS卵巢顆粒細胞凋亡的影響及其作用機制1. PCOS婦女與對照組卵巢顆粒細胞凋亡的對比應用流式細胞術對23例PCOS婦女以及30例對照卵巢顆粒細胞檢測發(fā)現(xiàn),PCOS組卵巢顆粒細胞凋亡率明顯高于對照組(P0.05)。2. PDCD4表達沉默前后PCOS卵巢顆粒細胞凋亡的對比為了進一步證實PDCD4對于卵巢顆粒細胞促凋亡作用,通過PDCD4-SiRNA轉(zhuǎn)染卵巢顆粒細胞24h后發(fā)現(xiàn)其凋亡率明顯降低(P0.05),TUNEL分析也發(fā)現(xiàn)凋亡細胞數(shù)量明顯減少。3. PDCD4表達沉默前后PCOS卵巢顆粒細胞凋亡通路信號分子表達變化為了確定PDCD4表達沉默對于凋亡通路關鍵信號分子的影響,通過Western blot發(fā)現(xiàn),DCD4-SiRNA轉(zhuǎn)染卵巢顆粒細胞24h后Bax表達明顯降低,Bcl-2表達明顯升高,Bax/Bcl-2比值明顯降低。結(jié)論1. PDCD4在PCOS婦女中出現(xiàn)高表達,其中肥胖亞組PDCD4表達增強尤為顯著。2. PDCD4在PCOS婦女中的高表達與其肥胖指數(shù)、胰島素抵抗、脂代謝紊亂密切相關,是PCOS婦女發(fā)病的獨立風險因素。二甲雙胍治療后PDCD4表達降低。3. PDCD4高表達促進PCOS婦女卵巢顆粒細胞凋亡,其作用途徑主要是通過Bax/Bcl-2凋亡信號通路。
[Abstract]:The background of polycystic ovary syndrome (Polycystic ovary, syndrome, PCOS) is a disease of endocrine and metabolic abnormalities common in women of childbearing age, chronic anovulation, Kaohsiung and hormone level of polycystic ovaries as the main feature. The common clinical manifestations with infertility, hirsutism, menstrual disorders and obesity and its complications including II the incidence of diabetes, cardiovascular diseases,.PCOS female obesity rate was 47%, significantly higher than the general population, and the degree of obesity and insulin resistance (Insulin resistance IR) is highly relevant. A comparative study of women in obese and non obese type PCOS, obesity is more severe metabolic disorder in obese women type PCOS, obesity increased degree of IR. Blocking obesity, IR, break the vicious spiral metabolism between the block of the PCOS process, improve endocrine abnormalities and restore ovulation is important. Women of childbearing age eggs The bubble is influenced by a variety of hormones in the course of its development, the development of mature follicle experience, eventually ovulation. Each menstrual cycle only a mature follicle ovulation, the follicle atresia in different periods. A large number of studies show that follicular atresia associated with granulosa cell apoptosis in PCOS women. The apoptosis of ovarian granulosa cells increased significantly (.PDCD4 programed cell death 4) is the glucose and lipid metabolism related genes discovered in recent years and promoting apoptosis gene, the gene can significantly improve insulin sensitivity, inhibit the development of obesity, promote lipid metabolism, inhibit the apoptosis of.PDCD4 cells in PCOS in women whether there is abnormal expression which further leads to the disorder of glucose and lipid metabolism, enhance the apoptosis of granulosa cells that will help to reveal the PCOS metabolism and follicular development disorder pathogenesis, has important clinical significance in the occurrence and development of PCOS The expression characteristics of righteousness. The aim of this study is obese and non obese women with PCOS and controls the peripheral blood PDCD4, PDCD4 and PCOS on women's body mass index (Body mass, index, BMI), insulin resistance and lipid metabolism disorder correlation; through the study of effect of PDCD4 on apoptosis of ovarian granulosa cells in women with PCOS. To explore the molecular mechanism of PCOS follicular development disorder, provide a new approach for the treatment of PCOS ovulation induction therapy; metformin improves metabolic disorders to further observe the expression of PDCD4 gene after PDCD4 and PCOS, a clear correlation between Xie Yichang, PCOS provides a theoretical basis for elucidating the pathogenesis of PCOS, and provide experimental basis for individualized treatment. 1. through the expression of PDCD4 in PCOS state and control women in the crowd, clear PDCD4 in patients with PCOS expression characteristics and PCOS expression analysis of PDCD4 2.; Women BMI, correlation between insulin resistance and lipid metabolism parameters; the expression detection of PDCD4 metformin improves insulin sensitivity, the expression of PDCD4 and PCOS to clarify the disorder of endocrine and metabolic correlation; 3. to investigate the effects of PDCD4 expression on PCOS in women with ovarian particle cell apoptosis and its mechanism. Methods 1. cases were collected in the treatment of reproductive hospital Shandong University affiliated PCOS women in 77 cases, the diagnosis of PCOS was based on the diagnostic criteria of PCOS China; the other normal menstrual cycle, for male or tubal factors were not pregnant women as a control group of 67 cases; all subjects excluded Cushing syndrome, thyroid dysfunction, androgen secretion of tumor and type I, II type I diabetes 3 months before entering the group did not receive any medical or surgical treatment; according to the BMI PCOS and the control group were divided into obese women The sub group (more than 25kg/m2) and non obesity subgroup (25kg/m2).2. Real time RT-PCR real-time fluorescence quantitative detection and separation of all patients peripheral blood mononuclear cells, RNA extraction, Real time using real-time fluorescence quantitative RT-PCR detection of PCOS pregnant women and control the expression of.3. in ovarian granulosa cells group PDCD4 mRNA separation in ultrasound guided. Transvaginal aspiration of follicular oocyte, follicle fluid aspirated found OCCC under stereomicroscope, the follicular fluid after picking eggs quickly transferred to the laboratory for granule cell washing and purification.4. Small interfering RNA (SiRNA) transfected with PDCD4-SiRNA sequence design, the use of PCOS women's ovarian granulosa cell purification liposome transfection separation at the same time after application and PDCD4 sequence; non homologous sequence as negative control PDCD4-SiRNA PCOS women's.5. detection by flow cytometry and the control group of ovarian granulosa cell division The purified V-FITC and PI immediately with Annexin (propidium iodide) apoptosis detection kit staining by flow cytometry analysis of ovarian granulosa cell apoptosis rate of.6. TUNEL was detected in women with PCOS of ovarian granulosa cells after separation of PDCD4-SiRNA transfected by TUNEL staining, apoptosis detection kit by PDCD4-SiRNA microscope before and after transfection and purification of apoptosis of ovary the changes of.7. Western blot in granulosa cells of ovarian granulosa cells in women with PCOS detection after separation of PDCD4-SiRNA transfected cells was extracted by confocal laser, determination of protein concentration followed by SDS-PAGE electrophoresis, transmembrane, antibody binding, scotograph, observe the PDCD4-SiRNA expression of.8. before and after transfection was ovarian granulosa cell apoptosis signal pathway of Bax and Bcl-2 analysis all data are used SPSS statistical analysis software, the measurement data to mean + standard deviation table Show, count data expressed in numerical and percentage; by one-way ANOVA, t test, Spearman regression analysis and multiple correlation factors; all were two-sided hypothesis test, P0.05 was considered statistically significant. As a result, PDCD4 in PCOS women and according to population in the different expression of 1.PCOS patients and the controls comparison of estradiol in PCOS patients clinical characteristics (E2), total testosterone (TT), luteinizing hormone (LH), fasting insulin (Insulin 0), oral glucose insulin after two hours (120 Insulin) and homeostasis model assessment of insulin resistance index (HOMA-IR) was significantly higher than the control group, the difference was statistically significant (P0.05; PCOS) women's follicle stimulating hormone (FSH) was significantly lower than the control group, the difference was statistically significant (P0.05); two groups of age, BMI, thyroid stimulating hormone (TSH), prolactin (PRL), fasting blood glucose (Glucose0), oral Two hours after the blood glucose (120 Glucose), islet P cell function index (HOMA-p), total cholesterol (TC), triglyceride (TG), low density lipoprotein (LDL) and high density lipoprotein (HDL) had no significant difference with the control group of women.2. PCOS in obese and non obese subgroup comparison of clinical characteristics PCOS women with obesity subgroup E2, TT, LH, Insulin 0, Insulin 120, Glucose 120 and HOMA-IR were significantly higher than the control obese subgroups, with statistical difference (P0.05); FSH was significantly lower than the control group, the difference was statistically significant (P0.05); two sub groups of age, BMI, TSH, PRL, Glucose 0. HOMA- TC, TG, beta, LDL and HDL had no statistically significant differences between.PCOS women and non obese subgroups TSH, E2, TT and LH were significantly higher than the control in non obese subgroups, with statistical difference (P0.05); FSH was significantly lower than the control group, the results have statistical difference (P0.05); two sub groups of age, BMI. PRL Insulin, 0, Insulin 120, Glucose 0, Glucose 120, HOMA-IR, TC, TG, HOMA- beta, LDL and HDL had no significant difference between the control group and the group.3. PCOS women and obese and non obese subgroups of PDCD4 expression by comparing with the control group of women with PCOS disease of peripheral blood mononuclear cells (PBMCs) real time quantitative PCR PDCD4 mRNA analysis found that the expression of PDCD4 in PCOS in women was higher than the control group, the difference was statistically significant (P0.05); PCOS women obesity subgroup PDCD4 expression was significantly higher than the control in obese subgroups, with significant statistical difference (P0.01); PCOS women and non obese control between the subgroups of PDCD4 expression had no significant difference. Two, the expression of PDCD4 and PCOS pregnant women and control group lipid metabolism correlation analysis and clinical significance of the expression of 1.PDCD4 and body mass index (BMI) has a significant correlation with the expression of PDCD4 BMI between PCOS women (r=0.52, P0.0001), for The control group the expression of PDCD4 PDCD4 and BMI.2. had no correlation with Insulin 0, Insulin 120, Glucose 120 PDCD4 expression correlation with insulin resistance in women with PCOS, HOMA-IR and HOMA- beta has significant correlation (r=0.34,0.31,0.29,0.36,0.25; P0.01 or 0.05); for the control group the expression of PDCD4 and insulin resistance index without any correlation to IR PCOS. Patients treated with metformin treatment for three months, according to the PDCD4 PBMCs mRNA real-time fluorescence quantitative PCR detection showed that PDCD4 expression was significantly reduced after metformin treatment, the difference was statistically significant (P0.05).3. PDCD4 expression had a significantly positive correlation with the expression of TG PDCD4 PCOS women's relationship with lipid metabolism in (r=0.39; P0.001), the expression of PDCD4 has significant negative correlation with HDL (r=-0.41; P0.001), the control group PDCD4 expression and lipid metabolism index did not Association of women with PCOS risk analysis by multi factor regression analysis showed that the expression of any correlation between.4. PDCD4 E2 and Insulin 120 after correction, the expression of PDCD4 in women with PCOS disease are independent risk factors (relative risk, 1.318; 95% Ci, 1.021-1.703, P0.05), shows that the high expression of PDCD4 increased the occurrence of with the development of PCOS, which reflects the important risk factors of the disease. Three women with PCOS, the expression of PDCD4 effect on apoptosis of ovarian granulosa cells PCOS and its mechanism of 1. PCOS women and control group comparison of ovarian granulosa cell apoptosis by flow cytometry in 23 cases of PCOS and 30 cases of women were detected ovarian granulosa cell apoptosis, PCOS group of ovarian granulosa cells was significantly higher than that of control group (P0.05).2. PDCD4 expression before and after contrast PCOS apoptosis of ovarian granulosa cells to further confirm that PDCD4 for ovarian fine particles The cell apoptosis of ovarian granulosa cells by PDCD4-SiRNA transfection, 24h showed that the apoptosis rate was significantly lower (P0.05), TUNEL analysis also found that the number of apoptotic cells decreased the expression of PDCD4.3. in order to determine the effects of PDCD4 gene silencing on key signaling molecules in apoptosis pathway of PCOS expression of granulosa cell apoptosis signal pathway by Western blot before and after the silence. DCD4-SiRNA found that transfection of 24h in ovarian granulosa cells after Bax expression decreased, Bcl-2 expression was significantly increased, Bax/Bcl-2 ratio decreased significantly. Conclusion 1. PDCD4 in women with PCOS showed high expression in the expression of obesity subgroup PDCD4,.2. PDCD4 in PCOS is significantly enhanced in women with high expression of obesity index, insulin resistance, lipid metabolism is closely related disorder is an independent risk factor for women with PCOS pathogenesis. The decreased expression of PDCD4.3. PDCD4 high expression increased after metformin treatment The apoptosis of ovarian granulosa cells in PCOS women is mainly through the pathway of Bax/Bcl-2 apoptosis signal.

【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

【參考文獻】

相關期刊論文 前3條

1 陳瑜;;瘦素受體Gln223Arg基因多態(tài)性與多囊卵巢綜合征胰島素抵抗及脂質(zhì)代謝的關系[J];臨床醫(yī)藥實踐;2011年12期

2 張靜;劉旗;;瘦素與腫瘤壞死因子α基因多態(tài)性與多囊卵巢綜合征發(fā)病相關性研究[J];中國衛(wèi)生檢驗雜志;2010年12期

3 喬杰,陳詠健,朱馥麗,楊池蓀,毛文偉;多囊卵巢綜合征血漿瘦素的改變及相關影響因素的研究[J];中國婦產(chǎn)科臨床;2002年03期

，

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