二甲双胍调控miRNA-133b对多囊卵巢综合征患者卵丘颗粒细胞增殖和凋亡的影响

发布时间：2018-07-03 15:23

本文选题：多囊卵巢综合征 + 颗粒细胞　；参考：《广州医科大学》2017年硕士论文

【摘要】：目的多囊卵巢综合征(PCOS),发病率高达5%-10%,主要特点是稀发排卵或无排卵、高雄激素血症及超声下卵巢呈多囊样改变,目前PCOS已严重影响到女性的生殖与健康,但该病发病机制尚不明确,现认为PCOS发病与胰岛素抵抗和高胰岛素血症、免疫因子失衡、环境及心理因素改变等有关。已知卵丘颗粒细胞在卵泡发育成熟中起重要作用,PCOS患者卵泡发育和成熟障碍导致无排卵是其不孕的重要原因之一。近几年发现二甲双胍在改善PCOS患者胰岛素抵抗的同时也可有效改善PCOS患者的排卵的作用。有学者对PCOS患者血清中miRNA打谱研究发现,PCOS患者血清中miRNA-133b表达下降,而本课题组近年来对miRNA-133b的相关研究发现,miRNA-133b在人类MI期卵母细胞中呈高表达;在小鼠卵母细胞中发现miRNA-133b可影响卵泡发育;在ICSI患者卵丘颗粒细胞体外培养研究发现,miRNA-133b可促进颗粒细胞增殖,抑制颗粒细胞凋亡,且在颗粒细胞中miRNA-133b与caspase-3表达呈相反趋势。但本课题组未在PCOS疾病患者卵丘颗粒细胞中进行miRNA-133b与caspase-3表达的研究,也未对二甲双胍对PCOS疾病患者卵丘颗粒细胞中miRNA-133b与caspase-3表达的具体影响进行研究。由于二甲双胍影响卵泡生长发育的机制还不明确,所以探究二甲双胍是否通过干扰PCOS患者卵丘颗粒细胞中miRNA-133b的表达进而影响颗粒细胞增殖和凋亡状态,具有重要意义。方法收集2016年09月至2017年04月就诊于广州医科大学附属第三医院生殖医学中心,年龄在20-40岁,行IVF-ET治疗的非PCOS,PCOS及近三个月口服二甲双胍治疗的PCOS女性不孕症患者的卵泡液,分离提取其中颗粒细胞进行体外培养及如下研究:(1)采用光学显微镜观察人卵丘颗粒细胞体外培养过程中的生长情况,用CCK-8法检测细胞活性。(2)用Q-PCR法检测颗粒细胞体外培养过程中miRNA-133b及caspase-3的表达。(3)体外二甲双胍干预后观察颗粒细胞活性及生长状态,同时检测细胞中miRNA-133b及caspase-3的表达。结果1、正常状态下(二甲双胍未在体外干预)三组颗粒细胞(non-PCOS,PCOS,PCOS+metformin组)的生长状态、活性及细胞内miRNA-133b与caspase-3的表达情况(1)经光学显微镜观察发现,三组颗粒细胞在0h-192h体外培养过程中生长状态近似,无显著差异。(2)用CCK-8法检测细胞活性发现,整体上三组细胞生长趋势相同,与光学显微镜观察的生长状态结果近似。只是在72h和96h时PCOS组细胞的活性显著低于non-PCOS组(p0.05)。(3)Q-PCR结果显示,三组颗粒细胞在体外培养过程中其内miRNA-133b与caspase-3表达有显著差异(p0.05)。且miRNA-133b与caspase-3表达呈相反趋势。2、不同浓度二甲双胍干预后,三组颗粒细胞的生长状态、活性及细胞内miRNA-133b与caspase-3的表达情况(1)光学显微镜下观察发现:二甲双胍浓度≥50mmol/l可对细胞致死;二甲双胍浓度在10mmol/l-50mmol/l,可影响细胞生长,细胞内可见细小颗粒样空泡;二甲双胍浓度在0mmol/l-5mmol/l细胞生长正常。(2)CCK-8检测发现:二甲双胍浓度在1-20mmol/l时可提高颗粒细胞活性(p0.01),30-50mmol/l显著降低细胞活性(p0.01)。(3)Q-PCR结果显示,不同浓度二甲双胍对三组颗粒细胞内miRNA-133b与caspase-3表达有显著影响(p0.05),但三组细胞间RNA的表达无规律可循。3、1mmol/l二甲双胍干预后,三组细胞的生长状态、活性及细胞内miRNA-133b与caspase-3的表达情况(1)光学显微镜下观察发现,经1mmol/l二甲双胍浓度干预颗粒细胞后,PCOS组颗粒细胞在d6-d8天凋亡明显减轻。(2)CCK-8法检测细胞活性发现PCOS组颗粒细胞在72h和96h时活性增高,与non-PCOS组细胞间无差异(p0.05),但在细胞培养后期PCOS患者的颗粒细胞活性依旧低于non-PCOS组(p0.05)。(3)Q-PCR结果:整体上三组细胞中miRNA-133b与cleaved caspase-3表达呈相反趋势;具体的,non-PCOS组颗粒细胞中二者表达趋势较正常情况下相比不变,而PCOS组和PCOS+metformin组颗粒细胞中miRNA-133b表达在d8天表达下降(p0.05)。结论1、人卵泡液中颗粒细胞体外培养过程中,正常生长周期只有8天,生长状态在72h时最佳,这与患者的病种无关。2、miRNA-133b可能参与PCOS的发病机制。3、二甲双胍可能通过调控miRNA-133b的表达进而影响颗粒细胞的增殖和凋亡。4、二甲双胍对PCOS患者卵丘颗粒细胞增殖和凋亡状态影响更明显。
[Abstract]:Objective polycystic ovary syndrome (PCOS), with a high incidence of 5%-10%, mainly characterized by dilute ovulation or anovulatory, polycystic changes in the ovary of Kaohsiung with steroids and ultrasound. At present, PCOS has seriously affected the reproductive and health of women. However, the pathogenesis of the disease is not yet clear. It is believed that the pathogenesis of PCOS is with insulin resistance and hyperinsulinemia. Unbalance of immune factors and changes of environmental and psychological factors. The known cumulus granulocyte plays an important role in the development of follicle maturation. Anovulatory anovulatory is one of the important reasons for PCOS patients. Metformin has been found to improve insulin resistance in PCOS patients and improve PCOS in recent years. The role of miRNA in the serum of patients with PCOS showed that the expression of miRNA-133b in the serum of PCOS patients decreased, and the study of miRNA-133b in recent years found that miRNA-133b was highly expressed in human MI oocytes; in mouse oocytes, it was found that miRNA-133b could affect follicle development; in I, I was found in the oocytes of mice; In vitro culture of CSI patients with cumulus granulosa cells found that miRNA-133b could promote the proliferation of granulosa cells and inhibit the apoptosis of granulosa cells, and the expression of miRNA-133b and Caspase-3 in granulosa cells was opposite. However, the study group did not study the expression of miRNA-133b and Caspase-3 in the cumulus granulosa cells of the patients with PCOS disease, nor did the two a. The effect of guanidine on the expression of miRNA-133b and Caspase-3 in the cumulus granulosa cells of patients with PCOS disease is studied. The mechanism of metformin's influence on the growth and development of follicle is not clear. Therefore, it is explored whether metformin can interfere with the proliferation and apoptosis of granulosa cells by interfering with the expression of miRNA-133b in the cumulus granulosa cells of PCOS patients. Methods from 2016 09 months to 04 months in the reproductive medical center affiliated to the Third Affiliated Hospital of Guangzhou Medical University, the follicular fluid of non PCOS, PCOS and PCOS female infertility treated with IVF-ET, aged 20-40 years old, was treated with metformin for nearly three months, and the granulosa cells were isolated and extracted in vitro. The following studies are as follows: (1) the growth of human cumulus granulosa cells in vitro culture was observed by optical microscopy. Cell activity was detected by CCK-8 method. (2) the expression of miRNA-133b and Caspase-3 during the culture process of granulosa cells in vitro was detected by Q-PCR. (3) the activity and growth state of granulosa cells were observed by two metformin in vitro. The expression of miRNA-133b and Caspase-3 in the cells was detected. Results 1, the growth state of the three groups of granulosa cells (non-PCOS, PCOS, PCOS+metformin) under normal condition (non-PCOS, PCOS, PCOS+metformin), and the expression of miRNA-133b and Caspase-3 in the cells (1) were observed by optical microscopy. It was found that the three groups of granular cells were cultured in vitro in 0h-192h. There was no significant difference in growth state during the breeding process. (2) detection of cell activity by CCK-8 method found that the growth trend of three groups on the whole was the same as that observed by optical microscope. Only at 72h and 96h, the activity of cells in group PCOS was significantly lower than that in non-PCOS group (P0.05). (3) Q-PCR results showed that the three groups of granulosa cells were in body The expression of miRNA-133b and Caspase-3 in the external culture was significantly different (P0.05). And the expression of miRNA-133b and caspase-3 was opposite to.2. The growth state, activity of three groups of granular cells and the expression of miRNA-133b and Caspase-3 in different concentrations of metformin were observed at different concentrations. (1) the concentration of metformin was observed under the optical microscope. More than 50mmol/l could kill the cells. The concentration of metformin at 10mmol/l-50mmol/l could affect the cell growth, the cell could be seen in the cell, and the concentration of metformin in 0mmol/l-5mmol/l cells was normal. (2) CCK-8 detection showed that the concentration of metformin in 1-20mmol/l could increase the activity of granulosa cells (P0.01) and 30-50mmol/l significantly decreased. Cell activity (P0.01). (3) Q-PCR results showed that different concentrations of metformin had significant effects on the expression of miRNA-133b and Caspase-3 in the three groups of granulosa cells (P0.05), but the expression of RNA in the three groups could be followed by the prognosis of.3,1mmol/l metformin, the growth state, activity of the three groups and the expression of miRNA-133b and Caspase-3 in the cells (1) Under the optical microscope, it was found that after the concentration of 1mmol/l metformin interfered with granulosa cells, the apoptosis of PCOS group granulosa cells decreased obviously on D6-D8 days. (2) the activity of cell activity in PCOS group was increased when 72h and 96h were detected by CCK-8 method, and there was no difference between the cells of group non-PCOS and non-PCOS group (P0.05), but the particle size of PCOS patients in the later stage of cell culture was fine. The cell activity was still lower than that of the non-PCOS group (P0.05). (3) Q-PCR results: the expression of miRNA-133b and cleaved caspase-3 in the whole three groups of cells was opposite; specifically, the expression trend of the two in the granulosa cells of group non-PCOS was less than that in the normal condition, while the miRNA-133b expression in PCOS and PCOS+ metformin groups decreased in D8 days. 0.05) 0.05) conclusion 1, in vitro culture of granulosa cells in human follicular fluid, the normal growth cycle is only 8 days, the growth state is best at 72h, which is not related to the patient's disease.2, miRNA-133b may be involved in the pathogenesis.3 of PCOS, and metformin may affect the proliferation and apoptosis of granulosa cells by regulating the expression of miRNA-133b, and then.4, two a double. Guanidine had a more significant effect on the proliferation and apoptosis of cumulus granulosa cells in PCOS patients.
【学位授予单位】：广州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R711.75

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