【作者】 张娟；

【导师】 朱桂金；

【作者基本信息】 华中科技大学 ， 妇产科学， 2007， 博士

【摘要】 第一部分多囊卵巢综合征大鼠模型的建立目的:建立一种理想的多囊卵巢综合征(PCOS)动物实验模型。方法:选用23日龄雌性SD大鼠,实验组大鼠采用脱氢表雄酮(DHEA)硫酸钠盐制剂硫酸普拉睾酮钠9mg/100g/日+注射用水0.4ml皮下注射20天,对照组同期每日皮下注射0.4ml注射用水,连续20天。分别观察两组大鼠卵巢外观、重量、光镜HE染色形态学改变及透射电镜卵巢超微结构变化,测定血清睾酮(T)、雌二醇(E2)、孕酮(P)、催乳素(PRL)含量。结果:1.实验组大鼠卵巢重量显著高于对照组(P<0.01),肉眼可见卵巢表面较多囊性扩张的卵泡。2.光镜HE染色见实验组大鼠有较多囊性扩张的卵泡,卵泡颗粒细胞层变薄,卵泡膜细胞增生,较少有黄体形成。3.透射电镜观察见实验组大鼠卵巢颗粒细胞形态变形,呈梭形变,细胞核核形不整,核内染色质浓集。细胞间纤维增生,卵泡膜细胞层数增多,膜细胞内可见到脂滴。4.实验组大鼠血清T和E2浓度均显著高于对照组﹙P<0.05﹚。血清P浓度较对照组偏低,PRL浓度较对照组稍高,但差异无显著性(P>0.05)。结论:硫酸普拉睾酮钠诱导的雌性大鼠PCOS动物模型卵巢病理形态学改变及血内分泌改变与PCOS病人相似,是较理想的PCOS动物模型。第二部分多囊卵巢综合征大鼠卵泡发育障碍与颗粒细胞凋亡的相关性目的:探讨PCOS大鼠卵泡发育障碍与颗粒细胞凋亡的相关性。方法:采用硫酸普拉睾酮钠诱导大鼠PCOS模型,透射电镜及TdT介导的dUTP缺口末端标记(TUNEL)法检测大鼠卵巢颗粒细胞凋亡情况。结果:1.透射电镜显示,PCOS大鼠卵巢颗粒细胞表现出凋亡所特有的超微结构变化,细胞核核膜皱缩,核形不整,核内染色质浓缩,线粒体肿胀,线粒体嵴断裂甚至消失。在凋亡后期颗粒细胞内可见典型的半月形凋亡小体。2. TUNEL法检测PCOS组大鼠卵巢窦状卵泡颗粒细胞凋亡发生率较对照组明显增强(P<0.01),窦前卵泡颗粒细胞凋亡发生率两组无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未发现明显凋亡征象。结论:PCOS大鼠卵巢窦状卵泡颗粒细胞凋亡明显增强,这可能是PCOS卵巢中窦状卵泡发育停滞的原因之一。第三部分TRAIL及其受体DR4、DcR1在多囊卵巢综合征大鼠卵泡发育中的作用目的:探讨TRAIL(肿瘤坏死因子相关凋亡诱导配体)及其受体DR4(死亡受体4)、DcR1(诱骗受体1)在PCOS大鼠卵泡发育中的作用。方法:采用硫酸普拉睾酮钠诱导大鼠PCOS模型,免疫组化染色、RT-PCR分析及Western blot分析观察TRAIL及其受体DR4、DcR1在PCOS大鼠卵巢颗粒细胞的表达情况。结果:1.免疫组化结果显示,PCOS组大鼠卵巢窦状卵泡颗粒细胞TRAIL蛋白的表达较对照组明显增强(P<0.05),窦前卵泡颗粒细胞TRAIL蛋白的表达两组无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未见TRAIL蛋白表达。RT-PCR及Westernblot分析显示,PCOS组大鼠卵巢颗粒细胞TRAILmRNA及TRAIL蛋白的表达较对照组明显增强(P<0.01)。2.免疫组化结果显示,两组大鼠窦前卵泡和窦状卵泡颗粒细胞DR4蛋白的表达无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未见DR4蛋白表达。RT-PCR及Westernblot分析显示,两组大鼠卵巢颗粒细胞DR4 mRNA及DR4蛋白的表达无显著性差异(P>0.05)。3.免疫组化结果显示,PCOS组大鼠卵巢窦状卵泡颗粒细胞DcR1蛋白的表达较对照组明显减弱(P<0.01),窦前卵泡颗粒细胞DcR1蛋白的表达两组无显著性差异(P>0.05),两组卵巢始基卵泡颗粒细胞未见DcR1蛋白表达。RT-PCR及Westernblot分析显示,PCOS组大鼠卵巢颗粒细胞DcR1mRNA及DcR1蛋白的表达较对照组明显减弱(P<0.01)。结论:TRAIL及其受体DR4、DcR1在PCOS大鼠卵泡发育颗粒细胞凋亡中发挥了调控作用。TRAIL及其受体DR4、DcR1在PCOS颗粒细胞的表达异常可能是导致PCOS卵泡发育障碍的机制之一。第四部分Caspase3在多囊卵巢综合征大鼠颗粒细胞的表达目的:探讨TRAIL信号途径在PCOS大鼠卵巢颗粒细胞诱导凋亡的最终效应阶段。方法:采用硫酸普拉睾酮钠诱导大鼠PCOS模型,Westernblot分析及RT-PCR分析观察Caspase3(半胱氨酸天门冬氨酸蛋白酶3)在PCOS大鼠卵巢颗粒细胞的表达情况。结果:1.Westernblot分析显示,PCOS组大鼠卵巢颗粒细胞活性Caspase3蛋白的表达较对照组明显增强(P<0.01)。2. RT-PCR分析显示,PCOS组大鼠卵巢颗粒细胞Caspase3 mRNA的表达较对照组明显增强(P<0.01)。结论:TRAIL信号途径在PCOS大鼠颗粒细胞诱导的凋亡可能最终经过了Caspase级联,引起Caspase3的激活导致细胞凋亡。更多还原

【Abstract】 Part 1 Establishing a rat model of polycystic ovary syndromeObjective: To establish an animal model of polycystic ovary syndrome(PCOS)．Methods: 23-day-old female Sprague-Dawley rats were choosed in this research．The ratsin experiment group were injected daily S.C. (Subcutaneous injection) with SodiumPrasterone Sulfate 9mg/100g for 20 days．The rats in control group were injected withwater for injection．The ovarian aspect，weight，morphology were observed，the ovarianultrastructure were examined with transmission electron microscope(TEM)，and the serumtestosterone(T)，estradiol(E2)，progesterone(P)，prolactin(PRL) levels were assessedbetween each group．Results:1.In experiment group rats，the ovarian weight were significantly higher than that incontrol group（P＜0.01）,and there were many cystic follicles on the surface of theovary．2. Haematoxylin eosin staining of light microscope showed there were many cystic folliclesin the experiment group rats．The granular cell layer get attenuated，theca cellsproliferated and there were less corepus luteum forming．3. In experiment group rats，TEM showed that the granulosa cells were fusiform shape，nucleus were irregularly and intranuclear chromatin condensed．The fiber between cellincremented，theca cells layer increased，and there were lipid droplet in the theca cells．4. The serum T and E2 levels of rats in experiment group were significantly higher thanthose of control group（P＜0.05）．The serum P and PRL levels of rats in experiment group were similar to those of control group（P＞0.05）．Conclusion: The morphologic and endocrinological changes in the ovarian of the SodiumPrasterone Sulfate induced PCOS rat model were similar to those of PCOS patients．It wasproved to be an ideal PCOS animal model．Part 2 The relationship between follicle development arrested andgranulosa cells apoptosis in polycystic ovary syndrome ratsObjective：To investigate the relationship between follicle development arrested andgranulosa cells apoptosis in PCOS rats．Methods: Using Sodium Prasterone Sulfate induced PCOS rat model，the apoptosis of thegranulose cells were observed by TEM and TdT-mediated dUTP-biotin nickend-labeling(TUNEL)．Results:1. The ultrastructure of the granulose cells in PCOS rats displayed apoptoticcharacteristics：karyotheca of nucleus shrinkaged，karyotype aligned irregularly，intranuclear chromatin condensed，mitochondria swelled，mitochondrial cristae broke ordisappeared，and there were representative crescent-shaped apoptotic bodies in thegranulosa cells on the later stage of apoptosis．2. The apoptosis was detected by TUNEL．The incidence of apoptosis in granulosa cells ofantral follicles was significantly higher in PCOS rats than that in control group (P<0.01).The incidence of apoptosis in granulosa cells of preantral follicles was similar betweenthe two groups (P>0.05).There were no signs of apoptosis in granulosa cells ofprimordial follicles in either group．Conclusion: The incidence of apoptosis in granulosa cells of antral follicles wassignificantly higher in the PCOS rats．This may be one of the reason that induced arrested growth of antral follicles in the PCOS．Part 3 The role of tumor necosis factor-related apoptosis-inducingligand，death receptor 4 and decoy receptor 1 during follicle developmentin polycystic ovary syndrome ratsObjective：To investigate the role of necosis factor-related apoptosis-inducingligand(TRAIL)，death receptor 4(DR4) and decoy receptor 1(DcR1) during follicledevelopment in polycystic ovary syndrome rats．Methods：Using Sodium Prasterone Sulfate induced PCOS rat model，detected theexpression of TRAIL，DR 4 and DcR1 in granulosa cells within polycystic ovary syndromerats through the immunhistochemical staining，reverse transcription polymerase chainreaction (RT-PCR) analysis and western blot analysis．Results：1. Immunohistochemical analyses showed，the expression of TRAIL protein in granulosacells were significantly stronger in antral follicles from the PCOS rats than in thosefrom the control rats(P<0.05)，the expression of TRAIL protein in granulosa cells werenot significantly difference in preantral follicles between the PCOS rats and the controlrats(P>0.05)，there were no positive staining of TRAIL protein in granulosa cells ofprimordial follicles in either group．RT-PCR and western blot analysis showed，theexpression of TRAIL mRNA and TRAIL protein were significantly increased ingranulosa cells from the PCOS rats than in those from the control rats(P<0.01)．2. Immunohistochemical analyses showed，the expression of DR4 protein in granulosacells were not significantly difference in preantral and antral follicles between thePCOS rats and the control rats (P>0.05)，there were no positive staining of DR4 protein in granulosa cells of primordial follicles in either group．RT-PCR and western blotanalysis showed，the expression of DR4 mRNA and DR4 protein were not significantlydifference in granulosa cells between the PCOS rats and the control rats(P>0.05)．3. Immunohistochemical analyses showed，the expression of DcR1 protein in granulosacells were significantly weaker in antral follicles from the PCOS rats than in those fromthe control rats(P<0.01)，the expression of DcR1 protein in granulosa cells were notsignificantly difference in preantral follicles between the PCOS rats and the controlrats(P>0.05)，there were no positive staining of DcR1 protein in granulosa cells ofprimordial follicles in either group．RT-PCR and western blot analysis showed，theexpression of DcR1 mRNA and DcR1 protein were significantly decreased in granulosacells from the PCOS rats than in those from the control rats(P<0.01)．Conclusion：TRAIL，DR4 and DcR1 played a role in regulation the apoptosis of granulosecells during follicle development in PCOS rats．The abnormal expression of TRAIL，DR4and DcR1 in granulosa cells may be one of the modulation mechanism that induced thefailure during follicle development in PCOS．Part 4 The expression of caspase3 in granulosa cells within polycysticovary syndrome ratsObjective：To investigate the finally effective stage of TRAIL signaling pathway duringapoptosis in granulosa cells within polycystic ovary syndrome rats．Methods：Using Sodium Prasterone Sulfate induced PCOS rat model，detected theexpression of caspase3 in granulosa cells within polycystic ovary syndrome rats through thewestern blot analysis and reverse transcription polymerase chain reaction (RT-PCR) analysis．Results：1. Western blot analysis showed，the expression of actived caspase3 protein weresignificantly increased in granulosa cells from the PCOS rats than in those from thecontrol rats(P<0.01).2. RT-PCR analysis showed，the expression of caspase3 mRNA were significantlyincreased in granulosa cells from the PCOS rats than in those from the controlrats(P<0.01).Conclusion：TRAIL signaling pathway may be pass the caspase cascade finally during theapoptosis of granulosa cells in PCOS rats，it induced apoptosis by activating caspase3．更多还原