【作者】 张红媛；

【导师】 朱付凡；

【作者基本信息】 中南大学 ， 妇产科学， 2010， 博士

【摘要】 多囊卵巢综合症(polycystic ovary syndrome, PCOS)是引起年轻妇女月经不调、闭经和不孕最常见的原因,在育龄妇女的发病率约4%-8%,占无排卵性不孕病因的50%-70%,严重影响了年轻妇女的生活质量及精神状态。以往从遗传因素、下丘脑-垂体-性腺轴功能失调、卵巢及肾上腺功能异常、胰岛素抵抗、肥胖等方面对PCOS的病因进行了大量的研究,但确切病因及发病机制仍不清楚。近年来,炎症学说倍受关注,已有的临床资料表明PCOS患者亚临床慢性炎症因子水平升高,推测PCOS的发病机制可能与慢性低度炎症性疾病有关。IL-18是近几年发现的一种作用强大的前炎症细胞因子,诱导TNF-α产生,进一步诱导IL-6及CRP的合成。目前研究发现IL-18在炎症级联反应中具有中心地位,对机体的免疫和炎症反应有非常重要的调节作用。最近研究发现IL-18与PCOS有关,特别是PCOS妇女血清中IL-18水平显著升高,其升高的程度与雄激素水平正相关。说明IL-18可能参与了PCOS的发病过程。PCOS以高雄激素血症为特征,卵巢的病理改变是卵泡膜细胞的过度增殖、小卵泡的过度发育及不排卵。卵泡膜细胞控制卵泡的发育及闭锁,调节卵巢甾体激素的合成,是PCOS患者雄激素过多合成和分泌的主要来源,卵泡膜细胞功能的失调节将导致病理状态的发生。研究表明在卵巢卵泡膜细胞表面存在IL-18受体,说明IL-18可直接作用于卵泡膜细胞,并可能参与了卵泡的发育过程。但其对卵泡膜细胞功能的影响及其在PCOS发病过程中可能发挥的作用及机制目前尚不清楚。IL-18刺激效应的信号传导是通过与其受体的结合实现的。IL-18BP是一种能与IL-18高亲和力特异性结合的蛋白因子,它通过与IL-18竞争结合而阻止IL-18与其受体结合,从而有效地抑制IL-18的生物学活性。因此,我们通过体外培养原代卵泡膜细胞,在体外建立无血清原代细胞培养模型来研究IL-18对卵泡膜细胞功能的影响及其可能的作用机制,并进一步探讨了IL-18BP对卵泡膜细胞的保护作用,这不仅可为进一步深入研究PCOS发生的炎症机制提供新的实验依据,也可为PCOS确立新一代治疗方案提供理论基础。本研究分四部分,第一部分采用牛新鲜卵巢进行原代卵泡膜细胞培养及鉴定,为后续实验提供物质基础；第二部分观察不同浓度IL-18,作用不同时间后卵泡膜细胞的增殖情况及类固醇激素合成的改变,观察IL-18在PCOS发病过程中的作用；第三部分通过研究IL-18对卵泡膜细胞中的细胞色素P45017酶、P450胆固醇侧链裂解酶和LH-R表达的影响,及卵泡大小不同其卵泡膜细胞IL-18R表达的不同,探讨IL-18影响卵泡膜细胞功能的可能的作用机制；第四部分探讨IL-18BP-Fc对IL-18诱导的卵泡膜细胞增殖、类固醇激素分泌的影响,观察IL-18BP-Fc是否可抑制IL-18的生物效应,为治疗PCOS提供新的思路。目的：培养出原代卵泡膜细胞,为后续研究提供物质基础。方法：获取新鲜的牛卵巢组织,用酶消化法培养出原代牛卵泡膜细胞,采用Percoll密度梯度离心法进行细胞纯化,运用细胞免疫化学染色方法分别检测细胞的角蛋白、波形蛋白及Ⅷ因子的表达,运用免疫荧光法检测细胞的CYP17A1的表达,从而达到鉴定卵泡膜细胞的目的。结果：镜下观察细胞生长良好,呈单层分布,细胞形态以短梭形为主,台盼蓝染色显示大于90%的卵泡膜细胞存活。细胞免疫化学染色结果显示细胞波形蛋白染色阳性,而角蛋白及Ⅷ因子染色阴性。免疫荧光结果显示细胞CYP17A1阳性表达,说明所培养的细胞为卵泡膜细胞。结论：原代牛卵泡膜细胞培养成功。目的：通过牛卵泡膜细胞原代培养体系,研究IL-18对卵泡膜细胞增殖及甾体激素合成及分泌功能的影响。方法：将相同数量的牛卵泡膜细胞分为对照组和研究组,其中对照组为正常培养细胞组,研究组为给予不同浓度的IL-18(10、30、50、100、300.500.1000 pg／ml)进行干预处理,各组分别于24h、48h及72h后收集细胞上清培养液,采用MTT法检测细胞增殖情况,采用放射免疫法检测细胞雄烯二酮及17羟孕酮的分泌。结果：不同浓度的IL-18干预相同时间后,浓度为10pg/ml-100pg/ml的研究组与对照组比较,细胞增殖的OD值、雄烯二酮与17羟孕酮的浓度的差异均无统计学意义(P>0.05)；浓度为300pg/ml-1000pg/ml的研究组与对照组比较,P<0.01,差异均有显著统计学意义；组间比较,P<0.01,差异有显著统计学意义。同一浓度的IL-18作用于卵泡膜细胞后不同时间(24h、48h、72h),各组OD值及雄烯二酮、17羟孕酮浓度进行比较,浓度100pg/ml以下的实验组,组间比较,P>0.05,差异均无统计学意义；浓度为300pg/ml以上的研究组,组间比较,P<0.01,差异均有显著统计学意义。结论：浓度<100pg/ml的IL-18对卵泡膜细胞的增殖及雄烯二酮、17羟孕酮的分泌无影响；浓度300pg/ml-1000pg/ml的IL-18可以诱导卵泡膜细胞的增殖,促进卵泡膜细胞雄烯二酮及17羟孕酮的分泌,且均呈剂量及时间依赖性。目的：通过牛卵泡膜细胞的原代培养体系,研究大、小卵泡IL-18受体(IL-18R)蛋白及mRNA表达的不同；研究IL-18对卵泡膜细胞中的P450c17酶、细胞色素P450胆固醇侧链裂解酶(P450scc)和LH-R蛋白表达的影响；研究IL-18对CYP17A1、CYP11A1和LH-R mRNA表达丰度的影响,探讨IL-18诱导PCOS的可能的发病机制,论证IL-18对女性排卵障碍产生影响的可能机理。方法：将相同数量的牛卵泡膜细胞随机分为对照组和研究组,对照组为正常培养细胞组,研究组则给予1000pg/ml的IL-18进行干预,两组均于72h后收集细胞上清培养液,采用Western blot方法检测卵泡膜细胞中P450c17酶、P450scc、LH-R蛋白表达的影响；采用荧光实时RT-PCR方法检测卵泡膜细胞中CYP17A1、CYP11A1、LH-R mRNA表达的影响。根据卵泡直径分为大卵泡组(8-20mm)和小卵泡组(<8mm),分别进行原代卵泡膜细胞培养,采用Western blot方法检测两组卵泡膜细胞中IL-18 R蛋白表达的不同,采用实时荧光RT-PCR检测两组卵泡膜细胞中IL-18R mRNA表达的不同。结果：与对照组相比,研究组卵泡膜细胞的P450c17酶.P450scc及LH-R蛋白表达均增加,差异均有显著统计学意义(P<0.01)。而且研究组卵泡膜细胞的CYP17A1、CYP11A1及LH-R的mRNA表达均显著增加,与对照组相比差异均有显著统计学意义(P<0.01)。小卵泡组的膜细胞IL-18R蛋白及mRNA表达水平与大卵泡组的膜细胞比较,均增高,差异有统计学意义(P<0.05)。结论：(1)小卵泡的膜细胞IL-18 R mRNA及蛋白表达均高于大卵泡的膜细胞。(2)IL-18可诱导卵泡膜细胞的雄激素合成过程中的关键酶P450c17酶、P450scc的表达增加,可能是引起卵泡膜细胞雄激素的分泌增高的机制。(3)IL-18可诱导卵泡膜细胞表面LH-R的表达增加。(4)IL-18可能通过与IL-18 R结合发挥作用,而诱导PCOS病理过程的发生。目的：IL-18BP-Fc是一种能与IL-18高亲和力特异性结合的蛋白因子,它通过与IL-18竞争结合而阻止IL-18与其受体结合,从而有效地抑制IL-18的生物学活性。本研究旨在观察IL-18BP-Fc对IL-18诱导卵泡膜细胞增殖及甾体激素分泌的影响,为PCOS的治疗提供新的思路。方法：取相同数量的卵泡膜细胞,随机分为四组：①对照组；②IL-18组；③IL-18BP-Fc组;④IL-18+IL-18BP-Fc组。其中第③组和第④组按IL-18BP-Fc浓度不同分为实验1组(1ng/ml)、实验2组(10ng/ml)、实验3组(100ng/ml)及实验4组(1000ng/ml)。第④组预先用IL-18BP-Fc预处理30分钟后,再加入1000pg/ml的IL-18。各组分别于4h、8h、16h及24h后收集细胞上清液进行检测。MTT法观察细胞增殖情况,放射免疫法检测上清液中雄烯二酮及17羟孕酮的含量。结果：IL-18BP-Fc组与对照组相比,细胞增殖及激素分泌量差异无统计学意义(P>0.05),说明IL-18BP-Fc对卵泡膜细胞无毒性。IL-18BP-Fc浓度为1ng/ml的实验组与IL-18组比较细胞增殖率及激素分泌量无明显差异,P>0.05,并且随着作用时间的延长,无明显变化。IL-18BP-Fc浓度为10ng/ml-1000ng/ml的实验组与IL-18组比较,细胞增殖率及激素分泌量均显著下降,P<0.05,组间比较均有统计学意义(P<0.05)。随着作用时间的延长抑制作用逐渐显著,16h时抑制作用达高峰(r=0.959,P<0.05)。浓度为1000ng/ml的IL-18BP-Fc作用16小时后,可使IL-18诱导卵泡膜细胞增殖及激素分泌的作用降低到约10%的水平,与对照组相比差异无统计学意义。24h时抑制作用明显减弱。结论：一定剂量的1L-18BP-Fc能够抑制IL-18对卵泡膜细胞的刺激效应;IL-18BP-Fc对IL-18的抑制效应呈时间及剂量依赖性；IL-18BP-Fc可能对卵泡膜细胞无毒性,且半衰期长。更多还原

【Abstract】 Polycystic ovary syndrome (PCOS) is the most common cause of oligomenorrhea, amenorrhea, and anovulatory infertility, and appears to be one of the most common endocrinopathy affecting women in their reproductive years. These patients are at higher risk of developing infertility, dysfunctional uterine bleeding, and a number of metabolic disorders. The prevalence of PCOS in reproductive-aged women is estimated to be about 4-8%, and account about 50-70% of the anovulatory infertility, which have greatly influenced the quality of life and mental status of young women. Although a series of study have carried out on the etiopathogenisis of PCOS, such as genetic factors, functional disorders of HPO axis, disfunction of adrenal and ovarian enzymics, insulin resistance and obesity, the precise aetiology is still not known.There is accumulating evidence indicating chronic low-grade inflammation in PCOS, including increased levels of CRP, increased leukocyte count, and increased pro-inflammatory cytokines (i.e. IL-6 and IL-18). These data suggest that the pathogenesy of PCOS may be associated with chronic low-grade inflammation and activation of the innate immune system. IL-18 is a new proinflammatory cytokine, discovered recently that induces the production of TNF-a, which in turn promotes the synthesis of IL-6, and IL-6 regulates thesynthesis of CRP in liver. Interleukin-18 (IL-18), initially known as interferon-y inducing factor, is a member of the IL-1 cytokine family and has been found to play an important role in innate and acquired immunity and inflammation response. Recently, elevated IL-18 levels were found in serum from some PCOS women, and the IL-18 levels were positively correlated with androgen levels, but were inversily correlated with insulin sensitivity. It indicates that IL-18 may be a contributing factor linking the onset of PCOS.PCOS ovaries are characterized by the accumulation of small follicles 4-7mm in diameter, with hyperplasia of the theca interna layers and excessive production of androgens by these cells. Thecal cells control follicle growth and atresia, regulate ovarian seroidogenesis, and are recognized as one of the primary sources of excess androgen biosynthesis in women with PCOS. Dysregulation of the thecal cells compartment will encountered in pathological conditions such as PCOS and hyperthecosis which are characterized by thecal and stromal hyperplasia and steroidogenic hyperactivity. The IL-18 receptor has been confirmed to be exist in thecal cells, and may be participant in follicle development process, although the exactly influence is still unknown.Extracelllular IL-18 exerts its biological effects by binding the heterodimeric IL-18 receptor complex. IL-18-binding protein(IL-18BP) is a naturally occurring protein that binds and neutralizes IL-18. It can binds IL-18 with specificity and high affinity and blocks its biological activities. Therefore, in this study, we examined whether or not IL-18 and its receptor were implicated in the proliferation of thecal cells in culture. In addition, we also analyzed the possibly mechanism of the modulating effect, and the possible protect effect of IL-18BP to thecal cells. Our work will provide not only new experimental data for furthering the pathogenesis of inflammation in PCOS in depth but also theoretical basis for setting up new therapies.Our research is divided into four parts. In the first part, we used fresh bovine ovaries to cultivate original bovine thecal cells, so that there were enough thecal cells to investigate. In the second part, we investigate the alteration of proliferation and steroidogenesis of thecal cells with different concentration of IL-18 with different time, and analyse the function of IL-18 in the morbidity of PCOS. In the third part, we investigate the possible mechanism of IL-18 effecting thecal cells. In the last part, we studied the effects of IL-18BP-Fc on the proliferation and steroidogenesis induced by IL-18 on thecal cells. To study the molecular mechanism of inflammation in PCOS and offer helpful experiments data for the treatment of PCOS by IL-18BP. Objective:In order to investigate the effect of IL-18 on cell proliferation and steroidogenisis of thecal cells, we did original cultivation of bovine thecal cells.Methods:We obtained fresh bovine ovaries to cultivate original bovine thecal cells through enzyme digestion and Percoll density gradient centrifugation. Then we used immune cytochemistry and immunofluorescence to identify thecal cells.Results:The cells we cultured was in good condition, monolayer, and short fusiform shaped. About 90% of the cells we cultivated were alive. The results of immune cytochemistry showed vimentin positively stained while keratinose andⅧfactor negatively stained. Under fluorescence microscope, the thecal cells were positively stained with CYP17A1. The cells we cultivited was thecal cells.Conclusions:We have successfully cultivated original bovine thecal cells. Objective:In the present study, we investigated the cell proliferation and steroidogenisis on original cultured bovine thecal cells induced by IL-18.Methods:Original thecal cells were cultured in free serum DMEM/F12 medium, and treated with different concentration of IL-18(10、30、50、100、300、500、1000 ng/ml). Culture solution were collected after treated for 24h,48h and 72h, respectively. Then, cell proliferation was tested by MTT assay, androstenedione and 17-hydroxyprogesterone were detected with radio-immunity.Results:There were no statistically difference between the control group and the groups with IL-18 in concentration of 10ng/ml-100ng/ml (P>0.05). Compared with the control group, the OD value and androstenedione and 17-hydroxyprogesterone concentrations were significantly different with cells treated with IL-18 between 300ng/ml and 1000ng/ml(P<0.01); The OD value of IL-18 in concentration of 300ng/ml、500ng/ml and 1000ng/ml decreased significantly (P<0.01), and the androstenedione and 17-hydroxyprogesterone concentrations of them incerased significantly (P＜0.01), as the concentration of IL-18 upgraded.The OD value and androstenedione and 17-hydroxyprogesterone concentrations were compared between the control group and experimental groups after treated with IL-18(<100ng/ml) for 24、48 and 72h, respectively, there were no significantly difference (P>0.05). While OD value was significantly decreased and androstenedione and 17-hydroxyprogesterone concentration were significantly increased in the experimental groups with the concentration of IL-18 was>300ng/ml, as the time prolonged.Conclusions:There was no difference in the cell proliferation and androstenedione and 17-hydroxyprogesterone concentrations between control group and groups treated with IL-18 with concentration lower than 100ng/ml. With concentrations between 300ng/ml and 1000ng/ml,IL-18 could dose and time dependently induce cell preliferation and steroidgenesis of the thecal cell. Objective:The present study was to investigate the effect of IL-18 on the protein and mRNA expression of cholesterol side-chain cleavage enzyme(P450scc), cytochrome P450(P450c) 17-hydroxylase, and LH-R. We also investigate the differences of the expression of protein and mRNA of IL-18 R between big and small ovary follicles. The aim of this study was to demonstrate the mechanism of the IL-18 resulting in PCOS, and the possible pathyway to anovulation.Methods:Cattle thecal cells were treated with IL-18 with the concentration of 1000 pg/ml. The cultured media were collected after 72h for detection. P450scc, P450c17-hydroxylase and LH-R protein were detected by Western bolt. CYP11A1, CYP17A1 and LH-R mRNA were detected by RT-PCR.Based on surface diameter, the thecal cells were collected from small (<8mm) and large (8-20mm) follicles respectively. Then we used Western blot and RT-PCR to investigate the differences of protein and mRNA of IL-18R between the two groups.Results:Significantly increased of P450scc(P<0.01), P450c17-hydroxylase (P<0.01) and LH-R(P＜0.01) protein expression were observed in cattle thecal cells that was treated with IL-18. Also, IL-18 has caused an increased effect of mRNA expression of CYP17A1(P<0.01), CYP11A1(P<0.01) and LH-R(P<0.01). The expression of IL-18R protein(P<0.05) and mRNA(P<0.05) was significantly different between the big and small ovarian follicles.Conclusions:IL-18 can directly effect the thecal cells, include the increased testosterone secretion by inducing the expression of CYP17A1, CYP11A1 and LH-R in thecal cells. The results of the increased recruitment of small follicles and anovulation maybe related with the overexpression of IL-18 R on small follicles. All of these changes may related to the disruption of steroid hormone production and hyperandrogenism likely happend in PCOS. Objective:A naturally occurring protein, interleukin 18 binding protein (IL-18BP), which is normally present in serum can bind IL-18 and block its bioavailability and subsequent function. Therefore, to determine the effects of IL-18BP-Fc on proliferation and steroidogenesis of thecal cells induced by IL-18, experiments were conducted in the last part of our experiment.Methods:Original cultured thecal cells was induced by IL-18 (1000ng/ml) alone or in combination with IL-18BP-Fc in different concentrations (1ng/ml, 10ng/ml, 100ng/ml and 1000ng/ml). Then to collect the supernate for detection. MTT was used to elucidate the effects of IL-18BP-Fc on proliferation of thecal cells, and radioimmunity was used to detect the content of androstenedione and 17-hydroxyprogesterone in the supernate.Results:There was no difference between IL-18 group and group treated with IL-18BP-Fc with concentration of lng/ml in the cell proliferation and the concentrations of androstenedione and 17-hydroxyprogesterone. With concentrations between 10ng/ml and 1000ng/ml, IL-18BP-Fc could time dependently inhibit cell preliferation and steroidgenesis induced by IL-18, and to a top role at 16 hours. Otherwise, there was an evident dose-effect ralationship of IL-18BP-Fc on the thecal cell proliferation and androstenedione and 17-hydroxyprogesterone concentrations induced by IL-18. After treated with IL-18BP-Fc with concentration of 1000ng/ml for 16 hours, the depressive effect could achieve to about 90 percent, and no statistically significant with control group.Conclusions:The stimulating effect to thecal cells by IL-18 could be inhibit by IL-18BP-Fc and the depressive effect was time and dose dependently. The IL-18BP-Fc was avirulent to thecal cells and with a long half life.更多还原