PDCD4基因在多囊卵巢综合征患者中的表达及其相关性研究

发布时间：2018-03-28 17:31

本文选题：PDCD4　切入点：多囊卵巢综合征　出处：《山东大学》2016年博士论文

【摘要】：背景多囊卵巢综合征(Polycystic ovary syndrome,PCOS)是育龄期妇女常见的一种内分泌代谢异常性疾病,以慢性无排卵、高雄激素血症和卵巢多囊样改变为主要特征。常见的临床表现有不孕、多毛、月经紊乱以及肥胖等,其远期并发症包括Ⅱ型糖尿病、心血管疾病等。PCOS妇女肥胖的发病率为47%,显著高于普通人群,且肥胖程度与胰岛素抵抗(Insulin resistance,IR)呈高度正相关。对肥胖型和非肥胖型PCOS妇女的比较研究发现,肥胖型较非肥胖型PCOS妇女存在更严重的代谢紊乱,肥胖加重了IR程度。阻断肥胖、IR,打破代谢间的恶性循环,对阻断PCOS进程、改善内分泌异常及恢复排卵具有重要意义。育龄期妇女卵泡在其发育过程中受多种激素的影响,使卵泡经历发育、成熟、最终排卵。每一月经周期只有一个卵泡成熟排卵,其余卵泡则在不同时期闭锁。大量研究表明卵泡闭锁与颗粒细胞凋亡有关。在PCOS妇女中卵巢颗粒细胞凋亡明显增加。PDCD4(programed cell death 4)是近年来新发现的糖脂代谢相关基因和促凋亡调控基因,其基因缺失能够明显改善胰岛素敏感性,抑制肥胖症的发生,促进脂质代谢,抑制细胞凋亡。PDCD4在PCOS妇女体内是否存在着异常表达从而进一步导致其糖脂代谢紊乱、颗粒细胞凋亡增强,将有助于揭示PCOS代谢异常及卵泡发育障碍的发生机制,对明确PCOS的发生发展有着重要的临床意义。本研究拟通过测定肥胖型和非肥胖型PCOS妇女以及对照人群外周血PDCD4的表达特点,探讨PDCD4与PCOS妇女的体重指数(Body mass index,BMI)、胰岛素抵抗以及脂质代谢紊乱的相关性；通过研究PDCD4对PCOS妇女卵巢颗粒细胞凋亡的影响,探讨PCOS卵泡发育障碍的分子机制,为PCOS的促排卵治疗提供新的治疗思路；进一步观察应用二甲双胍改善代谢紊乱后PDCD4基因表达水平的变化,明确PDCD4与PCOS代谢异常的相关性,为阐明PCOS发生机制提供理论依据,为PCOS的个体化治疗提供实验依据。目的1.通过比较PDCD4在PCOS妇女以及对照人群中的表达状态,明确PDCD4在PCOS患者中的表达特点；2.分析PDCD4表达与PCOS妇女BMI、胰岛素抵抗以及脂代谢参数的相关性；应用二甲双胍改善胰岛素敏感性后检测PDCD4表达水平的变化,阐明PDCD4表达与PCOS内分泌代谢紊乱是否存在相关性；3.探讨PDCD4表达对PCOS妇女卵巢颗粒细胞凋亡的影响及其作用机制。方法1.病例收集选取在山东大学附属生殖医院就诊的PCOS妇女77例,PCOS的诊断依据中国PCOS诊断标准；另选择月经周期正常、因输卵管因素或男方因素就诊的不孕妇女67例为对照组；所有研究对象排除Cushing综合征、甲状腺功能障碍、分泌雄激素肿瘤和Ⅰ型、ⅡI型糖尿病,入组前3个月均未接受过任何药物或手术治疗；根据BMI分别将PCOS妇女及其对照组分成肥胖亚组(≥25kg/m2)以及非肥胖亚组(25kg/m2)。2.实时荧光定量Real time RT-PCR检测分离所有病例外周血单个核细胞,提取RNA,利用实时荧光定量Real time RT-PCR检测PCOS妇女及其对照组PDCD4 mRNA的表达情况。3.卵巢颗粒细胞分离在超声探头引导下,经阴道穿刺抽吸卵泡取卵,抽吸出的卵泡液在实体显微镜下找到卵冠丘复合物,将拣卵后的卵泡液迅速转移至实验室进行颗粒细胞的洗涤纯化。4. Small interfering RNA (SiRNA)转染设计PDCD4-SiRNA序列,利用脂质体转染分离纯化后的PCOS妇女卵巢颗粒细胞；同时应用与PDCD4序列非同源性序列作为PDCD4-SiRNA阴性对照。5.流式细胞术检测PCOS妇女及对照组卵巢颗粒细胞分离纯化后,立即用Annexin V-FITC和PI(propidium iodide)凋亡检测试剂盒染色,经流式细胞仪检测分析卵巢颗粒细胞凋亡率。6. TUNEL检测PCOS妇女卵巢颗粒细胞分离纯化后进行PDCD4-SiRNA转染,利用TUNEL凋亡检测试剂盒染色,经激光共聚焦显微镜观察PDCD4-SiRNA转染前后卵巢颗粒细胞凋亡变化情况。7. Western blot检测PCOS妇女卵巢颗粒细胞分离纯化后进行PDCD4-SiRNA转染,提取细胞蛋白测定蛋白浓度后依次行SDS-PAGE电泳,转膜,抗体结合,暗室显影,观察PDCD4-SiRNA转染前后卵巢颗粒细胞凋亡信号通路分子Bax和Bcl-2的表达变化情况。8.统计学分析所有数据均使用SPSS统计学软件包分析,计量资料以均数±标准差表示,计数资料以数值和百分比表示；统计分析采用单因素方差、t检验、Spearman相关以及多因素回归分析；所有假设检验均进行双侧检验,P0.05被认为差异有统计学意义。结果一、PDCD4在PCOS妇女与对照人群中的不同表达状态1.PCOS患者与对照组临床特点的比较PCOS患者雌二醇(E2)、总睾酮(TT)、黄体生成素(LH)、空腹胰岛素(Insulin 0)、口服葡萄糖两小时后胰岛素(Insulin 120)以及稳态模型评估胰岛素抵抗指数(HOMA-IR)明显高于对照组,结果具有统计学差异(P0.05)；PCOS妇女卵泡刺激素(FSH)明显低于对照组,结果具有统计学差异(P0.05)；两组间年龄、BMI、甲状腺刺激素(TSH)、催乳素(PRL)、空腹血糖(Glucose0)、口服葡萄糖两小时后血糖(Glucose 120)、胰岛p细胞功能指数(HOMA-p),总胆固醇(TC)、甘油三酯(TG)、低密度脂蛋白(LDL)和高密度脂蛋白(HDL)没有统计学差异。2. PCOS妇女与对照组肥胖和非肥胖亚组临床特点的比较PCOS妇女肥胖亚组E2、TT, LH、Insulin 0、Insulin 120、Glucose 120以及HOMA-IR明显高于对照肥胖亚组,具有统计学差异(P0.05);FSH明显低于对照组,结果具有统计学差异(P0.05)；两亚组间年龄、BMI、TSH、 PRL、Glucose 0、HOMA-β、TC、TG、LDL和HDL没有统计学差异。PCOS妇女非肥胖亚组TSH、E2、TT以及LH明显高于对照非肥胖亚组,具有统计学差异(P0.05);FSH明显低于对照组,结果具有统计学差异(P0.05)；两亚组间年龄、BMI、PRL、Insulin 0、Insulin 120、Glucose 0、Glucose 120、 HOMA-IR、HOMA-β、TC、TG、LDL和HDL没有统计学差异。3. PCOS妇女与对照组间及其肥胖和非肥胖亚组间PDCD4表达的比较通过对PCOS妇女与对照组病例外周血单个核细胞(PBMCs)中PDCD4 mRNA实时荧光定量PCR检测分析发现,PDCD4在PCOS妇女中的表达高于对照组,结果具有统计学差异(P0.05);PCOS妇女肥胖亚组PDCD4表达明显高于对照肥胖亚组,具有显著的统计学差异(P0.01);PCOS妇女与对照非肥胖亚组间PDCD4表达没有统计学差异。二、PDCD4表达与PCOS妇女及其对照组糖脂代谢的相关性分析及其临床意义1.PDCD4表达与体重指数(BMI)的相关性PCOS妇女中PDCD4表达与BMI具有显著的相关性(r=0.52,P0.0001),对于对照组PDCD4表达与BMI没有相关性。2. PDCD4表达与胰岛素抵抗的相关性PCOS妇女中PDCD4表达与Insulin 0、Insulin 120、Glucose 120、HOMA-IR和HOMA-β具有显著的相关性(r=0.34,0.31,0.29,0.36,0.25；P0.01或者0.05)；对于对照组PDCD4表达与胰岛素抵抗指标没有任何相关性。针对IR的PCOS患者给予口服二甲双胍三个月治疗,通过对其PBMCs中PDCD4 mRNA实时荧光定量PCR检测发现,PDCD4在二甲双胍治疗后其表达明显降低,结果具有统计学差异(P0.05)。3. PDCD4表达与脂代谢紊乱的相关性PCOS妇女中PDCD4表达与TG具有显著的正相关性(r=0.39；P0.001),PDCD4表达与HDL具有显著的负相关性(r=-0.41；P0.001),对于对照组PDCD4表达与脂代谢指标没有任何相关性。4. PDCD4表达与PCOS妇女发病风险的关联性分析通过多因素回归分析发现,校正E2和Insulin 120后,PDCD4表达对于PCOS妇女发病是独立的风险因素(相对危险系数,1.318；95%可信区间,1.021-1.703,P0.05),表明PDCD4的高表达加重了PCOS的发生和发展,是反映PCOS妇女发病的重要危险因素。三、PDCD4表达对PCOS卵巢颗粒细胞凋亡的影响及其作用机制1. PCOS妇女与对照组卵巢颗粒细胞凋亡的对比应用流式细胞术对23例PCOS妇女以及30例对照卵巢颗粒细胞检测发现,PCOS组卵巢颗粒细胞凋亡率明显高于对照组(P0.05)。2. PDCD4表达沉默前后PCOS卵巢颗粒细胞凋亡的对比为了进一步证实PDCD4对于卵巢颗粒细胞促凋亡作用,通过PDCD4-SiRNA转染卵巢颗粒细胞24h后发现其凋亡率明显降低(P0.05),TUNEL分析也发现凋亡细胞数量明显减少。3. PDCD4表达沉默前后PCOS卵巢颗粒细胞凋亡通路信号分子表达变化为了确定PDCD4表达沉默对于凋亡通路关键信号分子的影响,通过Western blot发现,DCD4-SiRNA转染卵巢颗粒细胞24h后Bax表达明显降低,Bcl-2表达明显升高,Bax/Bcl-2比值明显降低。结论1. PDCD4在PCOS妇女中出现高表达,其中肥胖亚组PDCD4表达增强尤为显著。2. PDCD4在PCOS妇女中的高表达与其肥胖指数、胰岛素抵抗、脂代谢紊乱密切相关,是PCOS妇女发病的独立风险因素。二甲双胍治疗后PDCD4表达降低。3. PDCD4高表达促进PCOS妇女卵巢颗粒细胞凋亡,其作用途径主要是通过Bax/Bcl-2凋亡信号通路。
[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.

【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R711.75

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