【摘要】：目的 多囊卵巢綜合征（Polycystic ovarian syndrome,PCOS）是臨床上最常見的引起生育期女性卵泡發(fā)育障礙的疾病。高雄激素血癥是PCOS患者最典型的癥狀。本研究探討睪酮對于體外培養(yǎng)顆粒細(xì)胞增殖，分泌，以及對細(xì)胞骨架蛋白的影響。探討血清中雄激素濃度過高引起卵泡發(fā)育障礙的可能原因和機制。為臨床研究不孕癥患者控制性超促排卵的治療方案，減少并發(fā)癥的發(fā)生提供理論基礎(chǔ)。 方法 1、體外培養(yǎng)小鼠卵巢顆粒細(xì)胞，并分別用HE染色和FSHR免疫熒光染色作細(xì)胞鑒定。 2、顆粒細(xì)胞分別與等量不同濃度的睪酮（空白對照組和實驗組：10－8，10－7，10－6，10－5M）作用，作用時間為24h，48h，72h。分別提取培養(yǎng)上清液并收集培養(yǎng)細(xì)胞作指標(biāo)測定。 3、MTT檢測不同濃度睪酮對顆粒細(xì)胞活性的影響。 4、酶聯(lián)免疫吸附實驗（ELISA）檢測上清液中AMH，VEGF,HIF1ɑ的含量。 5、實時定量聚合酶鏈反應(yīng)（RT-PCR）檢測細(xì)胞中AMHmRNA,VEGFmRNA,HIF1ɑmRNA的表達(dá)。 6、細(xì)胞骨架肌動蛋白（β-actin）的免疫熒光染色，細(xì)胞核DAPI染色，并于激光共聚焦顯微鏡下觀察。 結(jié)果 1、MTT檢測細(xì)胞活性：10－5M組的睪酮能夠抑制顆粒細(xì)胞生長，10－8－10－6M組的睪酮能夠促進(jìn)顆粒細(xì)胞增殖。 2、AMH和AMHmRNA的表達(dá):與空白對照組相比，睪酮作用24h，實驗組10-7M和10-6M組，AMH含量分別上升了68%(P＝0.02)和1.2倍(P＝0.001)；睪酮作用48h,10-7M和10-6M組AMH含量分別上升了1.2倍(P＝0.001)和1.76倍(P＝0.000).睪酮作用48h后，10-7M和10-6M組AMHmRNA表達(dá)量分別上升了58%(P＝0.001)和24%(P＝0.04)。 3、VEGF的含量和VEGFmRNA的表達(dá)：睪酮作用24h后，10-7M和10-6M組VEGF分別增加了1.5倍(P＝0.03)和1.05倍(P＝0.04)；睪酮作用48h后，10-7M和10-6M組VEGF分別增加了1.88倍(P＝0.009)和1.37倍(P＝0.005).睪酮作用48h后，實驗組VEGFmRNA表達(dá)量明顯增加。與空白對照組相比，10-7M和10-6M組分別增加了74%(P＝0.005)和1.74倍(P＝0.000）. 4.HIF1ɑ的含量和HIF1ɑmRNA的表達(dá)：與空白對照組相比，，睪酮作用24h時，10-7M和10-6M組HIF1ɑ分別增加了2.5倍（P＝0.005）和3.88倍（P＝0.000）.在睪酮作用48h時，10-7M和10-6M組HIF1ɑ分別增加了2.06倍（P＝0.001）和2.37倍（P＝0.000）。睪酮作用48h時后與對照組相比，10-7M和10-6M HIF1ɑmRNA表達(dá)尚未發(fā)現(xiàn)明顯變化（P＝0.09，P=0.07）。 4、實驗組睪酮（濃度分別為10-7M和10-6M）作用48h后，顆粒細(xì)胞骨架肌動蛋白（F-actin）的免疫熒光染色和細(xì)胞核DAPI染色，發(fā)現(xiàn)骨架肌動蛋白含量和細(xì)胞核形態(tài)無明顯變化。 結(jié)論 1、高濃度睪酮促進(jìn)AMH的分泌，可能是睪酮間接導(dǎo)致卵泡發(fā)育障礙的原因之一。 2、睪酮使顆粒細(xì)胞培養(yǎng)液中VEGF,HIF1ɑ的分泌水平增加，造成卵泡液局部相對缺氧的微環(huán)境，可能間接影響卵泡正常發(fā)育。 3、睪酮對顆粒細(xì)胞骨架蛋白總量和構(gòu)象無明顯影響。
[Abstract]:Objective polycystic ovary syndrome (Polycystic ovarian syndrome,PCOS) is the most common disease causing follicular dysplasia in women. Hyperandrogenemia is the most typical symptom in patients with PCOS. The aim of this study was to investigate the effects of testosterone on the proliferation, secretion and cytoskeletal proteins of cultured granulosa cells in vitro. To explore the possible causes and mechanism of follicular dysplasia caused by high serum androgen concentration. To provide a theoretical basis for the clinical study of the treatment of controlled hyperstimulation of ovulation and the reduction of complications. Methods 1. Granulosa cells of mouse ovary were cultured in vitro and identified by HE staining and FSHR immunofluorescence staining. (2) granulosa cells were treated with the same concentration of testosterone (blank control group and experimental group: 10-8 ~ 10-7 ~ 10-6 ~ (-5) M) for 24 h ~ 48 h ~ (-1) ~ 72 h. The supernatant was extracted and the cultured cells were collected and measured. The effects of different concentrations of testosterone on granulosa cell activity were detected by MTT assay. 4. Enzyme linked immunosorbent assay (ELISA) was used to detect the content of AMH,VEGF,HIF1 in supernatant. 5. Real-time quantitative polymerase chain reaction (RT-PCR) was used to detect the expression of AMHmRNA,VEGFmRNA,HIF1 mRNA. 6. Immunofluorescence staining of cytoskeleton actin (尾-actin) and nuclear DAPI staining were observed under confocal laser microscope. Results 1MTT assay showed that testosterone in 10-5 M group could inhibit granulosa cell growth, and testosterone in 10-8 ~ (-6) M group could promote granulosa cell proliferation. 2the expression of AMH and AMHmRNA: compared with the control group, the levels of AMH in the 10-7M and 10-6M groups increased by 68% (P0. 02) and 1. 2 times (Pn0. 001) respectively. The levels of AMH in 10-7M and 10-6M groups increased 1.2 times (P0. 001) and 1. 76 times (P0. 000), respectively. After treated with testosterone for 48 h, the expression of AMHmRNA increased by 58% (P0. 001) and 24% (P0. 04) in 10-7M and 10-6M groups, respectively. 3The content of VEGFmRNA and the expression of VEGFmRNA: after treated with testosterone for 24 hours, VEGF of 10-7 M and 10-6 M groups increased by 1.5 times (P0. 03) and 1. 05 fold (P0. 04), respectively. The VEGF of 10-7 M and 10-6 M groups increased 1.88 times (P0. 009) and 1. 37 times (P0. 005), respectively, after 48 h of testosterone treatment. After treatment with testosterone for 48 h, the expression of VEGFmRNA in the experimental group was significantly increased. Compared with the control group, 10-7M and 10-6M increased by 74% (P0. 005) and 1. 74 times (P0. 000), respectively. The content of 4.HIF1 and the expression of HIF1 mRNA: compared with the control group, the HIF1 of 10-7 M and 10-6 M groups increased by 2.5 times (P0. 005) and 3. 88 times (Pt0. 000) at 24 h after treatment with testosterone. The HIF1 of 10-7 M and 10-6 M groups increased 2.06 times (P0. 001) and 2. 37 times (P0. 000) respectively when treated with testosterone for 48 h. After treatment with testosterone for 48 h, the expression of HIF1 mRNA in 10-7 M and 10-6 M was not significantly changed compared with that in control group (P < 0.09 or P ~ (0.07). 4After the treatment of testosterone (10-7 M and 10-6 M) for 48 h, immunofluorescence staining and nuclear DAPI staining of actin (F-actin) in granulosa cytoskeleton were observed. It was found that there were no significant changes in actin content and nuclear morphology. Conclusion 1. High concentration of testosterone promotes the secretion of AMH, which may be one of the causes of follicular dysplasia caused by testosterone. (2) testosterone increased the secretion of VEGF,HIF1 in granulosa cell culture medium, resulting in a relatively anoxic microenvironment in the follicular fluid, which may indirectly affect the normal development of follicles. 3. Testosterone had no significant effect on the total amount and conformation of granulosa cytoskeleton protein.
【學(xué)位授予單位】：遼寧醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R711.6

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