本文選題：多囊卵巢綜合征 + 顆粒細胞　； 參考：《廣州醫(yī)科大學》2017年碩士論文

【摘要】：目的多囊卵巢綜合征(PCOS),發(fā)病率高達5%-10%,主要特點是稀發(fā)排卵或無排卵、高雄激素血癥及超聲下卵巢呈多囊樣改變,目前PCOS已嚴重影響到女性的生殖與健康,但該病發(fā)病機制尚不明確,現(xiàn)認為PCOS發(fā)病與胰島素抵抗和高胰島素血癥、免疫因子失衡、環(huán)境及心理因素改變等有關(guān)。已知卵丘顆粒細胞在卵泡發(fā)育成熟中起重要作用,PCOS患者卵泡發(fā)育和成熟障礙導致無排卵是其不孕的重要原因之一。近幾年發(fā)現(xiàn)二甲雙胍在改善PCOS患者胰島素抵抗的同時也可有效改善PCOS患者的排卵的作用。有學者對PCOS患者血清中miRNA打譜研究發(fā)現(xiàn),PCOS患者血清中miRNA-133b表達下降,而本課題組近年來對miRNA-133b的相關(guān)研究發(fā)現(xiàn),miRNA-133b在人類MI期卵母細胞中呈高表達;在小鼠卵母細胞中發(fā)現(xiàn)miRNA-133b可影響卵泡發(fā)育;在ICSI患者卵丘顆粒細胞體外培養(yǎng)研究發(fā)現(xiàn),miRNA-133b可促進顆粒細胞增殖,抑制顆粒細胞凋亡,且在顆粒細胞中miRNA-133b與caspase-3表達呈相反趨勢。但本課題組未在PCOS疾病患者卵丘顆粒細胞中進行miRNA-133b與caspase-3表達的研究,也未對二甲雙胍對PCOS疾病患者卵丘顆粒細胞中miRNA-133b與caspase-3表達的具體影響進行研究。由于二甲雙胍影響卵泡生長發(fā)育的機制還不明確,所以探究二甲雙胍是否通過干擾PCOS患者卵丘顆粒細胞中miRNA-133b的表達進而影響顆粒細胞增殖和凋亡狀態(tài),具有重要意義。方法收集2016年09月至2017年04月就診于廣州醫(yī)科大學附屬第三醫(yī)院生殖醫(yī)學中心,年齡在20-40歲,行IVF-ET治療的非PCOS,PCOS及近三個月口服二甲雙胍治療的PCOS女性不孕癥患者的卵泡液,分離提取其中顆粒細胞進行體外培養(yǎng)及如下研究:(1)采用光學顯微鏡觀察人卵丘顆粒細胞體外培養(yǎng)過程中的生長情況,用CCK-8法檢測細胞活性。(2)用Q-PCR法檢測顆粒細胞體外培養(yǎng)過程中miRNA-133b及caspase-3的表達。(3)體外二甲雙胍干預后觀察顆粒細胞活性及生長狀態(tài),同時檢測細胞中miRNA-133b及caspase-3的表達。結(jié)果1、正常狀態(tài)下(二甲雙胍未在體外干預)三組顆粒細胞(non-PCOS,PCOS,PCOS+metformin組)的生長狀態(tài)、活性及細胞內(nèi)miRNA-133b與caspase-3的表達情況(1)經(jīng)光學顯微鏡觀察發(fā)現(xiàn),三組顆粒細胞在0h-192h體外培養(yǎng)過程中生長狀態(tài)近似,無顯著差異。(2)用CCK-8法檢測細胞活性發(fā)現(xiàn),整體上三組細胞生長趨勢相同,與光學顯微鏡觀察的生長狀態(tài)結(jié)果近似。只是在72h和96h時PCOS組細胞的活性顯著低于non-PCOS組(p0.05)。(3)Q-PCR結(jié)果顯示,三組顆粒細胞在體外培養(yǎng)過程中其內(nèi)miRNA-133b與caspase-3表達有顯著差異(p0.05)。且miRNA-133b與caspase-3表達呈相反趨勢。2、不同濃度二甲雙胍干預后,三組顆粒細胞的生長狀態(tài)、活性及細胞內(nèi)miRNA-133b與caspase-3的表達情況(1)光學顯微鏡下觀察發(fā)現(xiàn):二甲雙胍濃度≥50mmol/l可對細胞致死;二甲雙胍濃度在10mmol/l-50mmol/l,可影響細胞生長,細胞內(nèi)可見細小顆粒樣空泡;二甲雙胍濃度在0mmol/l-5mmol/l細胞生長正常。(2)CCK-8檢測發(fā)現(xiàn):二甲雙胍濃度在1-20mmol/l時可提高顆粒細胞活性(p0.01),30-50mmol/l顯著降低細胞活性(p0.01)。(3)Q-PCR結(jié)果顯示,不同濃度二甲雙胍對三組顆粒細胞內(nèi)miRNA-133b與caspase-3表達有顯著影響(p0.05),但三組細胞間RNA的表達無規(guī)律可循。3、1mmol/l二甲雙胍干預后,三組細胞的生長狀態(tài)、活性及細胞內(nèi)miRNA-133b與caspase-3的表達情況(1)光學顯微鏡下觀察發(fā)現(xiàn),經(jīng)1mmol/l二甲雙胍濃度干預顆粒細胞后,PCOS組顆粒細胞在d6-d8天凋亡明顯減輕。(2)CCK-8法檢測細胞活性發(fā)現(xiàn)PCOS組顆粒細胞在72h和96h時活性增高,與non-PCOS組細胞間無差異(p0.05),但在細胞培養(yǎng)后期PCOS患者的顆粒細胞活性依舊低于non-PCOS組(p0.05)。(3)Q-PCR結(jié)果:整體上三組細胞中miRNA-133b與cleaved caspase-3表達呈相反趨勢;具體的,non-PCOS組顆粒細胞中二者表達趨勢較正常情況下相比不變,而PCOS組和PCOS+metformin組顆粒細胞中miRNA-133b表達在d8天表達下降(p0.05)。結(jié)論1、人卵泡液中顆粒細胞體外培養(yǎng)過程中,正常生長周期只有8天,生長狀態(tài)在72h時最佳,這與患者的病種無關(guān)。2、miRNA-133b可能參與PCOS的發(fā)病機制。3、二甲雙胍可能通過調(diào)控miRNA-133b的表達進而影響顆粒細胞的增殖和凋亡。4、二甲雙胍對PCOS患者卵丘顆粒細胞增殖和凋亡狀態(tài)影響更明顯。
[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.
【學位授予單位】：廣州醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R711.75

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