睾酮引起卵泡发育障碍的机制研究
发布时间：2018-12-07 07:23
【摘要】：目的 多囊卵巢综合征（Polycystic ovarian syndrome,PCOS）是临床上最常见的引起生育期女性卵泡发育障碍的疾病。高雄激素血症是PCOS患者最典型的症状。本研究探讨睾酮对于体外培养颗粒细胞增殖，分泌，以及对细胞骨架蛋白的影响。探讨血清中雄激素浓度过高引起卵泡发育障碍的可能原因和机制。为临床研究不孕症患者控制性超促排卵的治疗方案，减少并发症的发生提供理论基础。 方法 1、体外培养小鼠卵巢颗粒细胞，并分别用HE染色和FSHR免疫荧光染色作细胞鉴定。 2、颗粒细胞分别与等量不同浓度的睾酮（空白对照组和实验组：10－8，10－7，10－6，10－5M）作用，作用时间为24h，48h，72h。分别提取培养上清液并收集培养细胞作指标测定。 3、MTT检测不同浓度睾酮对颗粒细胞活性的影响。 4、酶联免疫吸附实验（ELISA）检测上清液中AMH，VEGF,HIF1ɑ的含量。 5、实时定量聚合酶链反应（RT-PCR）检测细胞中AMHmRNA,VEGFmRNA,HIF1ɑmRNA的表达。 6、细胞骨架肌动蛋白（β-actin）的免疫荧光染色，细胞核DAPI染色，并于激光共聚焦显微镜下观察。 结果 1、MTT检测细胞活性：10－5M组的睾酮能够抑制颗粒细胞生长，10－8－10－6M组的睾酮能够促进颗粒细胞增殖。 2、AMH和AMHmRNA的表达:与空白对照组相比，睾酮作用24h，实验组10-7M和10-6M组，AMH含量分别上升了68%(P＝0.02)和1.2倍(P＝0.001)；睾酮作用48h,10-7M和10-6M组AMH含量分别上升了1.2倍(P＝0.001)和1.76倍(P＝0.000).睾酮作用48h后，10-7M和10-6M组AMHmRNA表达量分别上升了58%(P＝0.001)和24%(P＝0.04)。 3、VEGF的含量和VEGFmRNA的表达：睾酮作用24h后，10-7M和10-6M组VEGF分别增加了1.5倍(P＝0.03)和1.05倍(P＝0.04)；睾酮作用48h后，10-7M和10-6M组VEGF分别增加了1.88倍(P＝0.009)和1.37倍(P＝0.005).睾酮作用48h后，实验组VEGFmRNA表达量明显增加。与空白对照组相比，10-7M和10-6M组分别增加了74%(P＝0.005)和1.74倍(P＝0.000）. 4.HIF1ɑ的含量和HIF1ɑmRNA的表达：与空白对照组相比，，睾酮作用24h时，10-7M和10-6M组HIF1ɑ分别增加了2.5倍（P＝0.005）和3.88倍（P＝0.000）.在睾酮作用48h时，10-7M和10-6M组HIF1ɑ分别增加了2.06倍（P＝0.001）和2.37倍（P＝0.000）。睾酮作用48h时后与对照组相比，10-7M和10-6M HIF1ɑmRNA表达尚未发现明显变化（P＝0.09，P=0.07）。 4、实验组睾酮（浓度分别为10-7M和10-6M）作用48h后，颗粒细胞骨架肌动蛋白（F-actin）的免疫荧光染色和细胞核DAPI染色，发现骨架肌动蛋白含量和细胞核形态无明显变化。 结论 1、高浓度睾酮促进AMH的分泌，可能是睾酮间接导致卵泡发育障碍的原因之一。 2、睾酮使颗粒细胞培养液中VEGF,HIF1ɑ的分泌水平增加，造成卵泡液局部相对缺氧的微环境，可能间接影响卵泡正常发育。 3、睾酮对颗粒细胞骨架蛋白总量和构象无明显影响。
[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.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.6
本文编号：2366813
[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.
【学位授予单位】：辽宁医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R711.6
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相关期刊论文 前2条
1 赵考考;高莉;薛凯;董丽;刁飞扬;崔毓桂;刘嘉茵;;雄激素诱导小鼠颗粒细胞凋亡及其机制探讨[J];江苏医药;2011年16期
2 刘颖;刘小强;董丽;刁飞扬;马翔;崔毓桂;刘嘉茵;;雄激素对卵巢颗粒细胞乳酸生成的作用[J];江苏医药;2013年12期
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