【作者】 司丽芳；

【导师】 范光丽；

【作者基本信息】 西北农林科技大学 ， 神经生物学， 2010， 博士

【摘要】 正常妊娠时,一些细胞因子通过复杂的神经-内分泌-免疫网络的相互调节作用,在下丘脑-垂体-性腺轴的表达有所变化,这些细胞因子受某些内外界因素的影响表达紊乱时,往往会引发各种妊娠疾病,严重时会造成妊娠终止。目前,调控妊娠的一些细胞因子在有机体的调控特性及其相互作用的机理还不是十分清楚。有资料显示,白细胞介素-18(Interleukin-18, IL-18)、胰岛素样生长因子-1(Insulin-like growth factor 1 ,IGF-1)和γ-干扰素(Interferon-gamma ,IFN-γ)、均参与妊娠调控,但IL-18、IGF-1和IFN-γ在妊娠中的相互调控作用还不太清楚。本研究采用免疫组化SP法对妊娠各期大鼠下丘脑-垂体-性腺轴中IGF-1和IL-18的分布及IFN-γ对IGF-1和IL-18表达的影响进行了研究,并采用图象分析系统对其表达进行了分析。采用荧光定量RT-PCR法分别检测了植入后期大鼠卵巢和子宫中IL-18mRNA和IGF-1mRNA的表达变化及阴道壁注射IFN-γ后对卵巢和子宫中IL-18和IGF-1表达的影响,用ELISA法检测了外周血中IL-18和IGF-1水平的变化,并结合显微及形态分析法进一步研究了不同剂量IFN-γ对妊娠的影响,以期为妊娠早期胚胎丢失机理增添神经生物学研究资料。研究结果如下:1. IGF-1和IL-18在各妊娠期大鼠下丘脑视前室周核、视上核、视前大细胞核、视前内侧核、视前外侧核、视交叉上核、弓状核等21个核团均有表达,分布范围较广;在腺垂体及神经垂体均有表达;在卵巢表达于黄体的粒性黄体细胞、生长卵泡、成熟卵泡;子宫内膜基质细胞、子宫腺上皮细胞、子宫壁平滑肌细胞、血管内皮中也有IGF-1和IL-18阳性产物分布。提示IGF-1和IL-18通过下丘脑-垂体-性腺轴的途径对妊娠发挥着重要的功能。2.IGF-1和IL-18在妊娠大鼠下丘脑-垂体-性腺轴的表达在妊娠各期存在一定的差异:如IGF-1在下丘脑主要核团胚胎植入前期、植入期、植入后期表达持续升高,妊娠中期表达较低,妊娠晚期表达明显高于妊娠中期,IL-18在下丘脑主要核团胚胎植入前期和植入期的表达不断下降,植入后期表达显著升高。说明两者的表达具有一定的时间程序和空间特性,也有各自的特点。IGF-1在胚胎的植入后期及妊娠晚期表达较多,说明IGF-1可能在胚胎的发育及分娩的启动方面具有重要的时间程序调控作用,同时表现了在下丘脑及垂体水平上参与妊娠的空间调控特性,而在卵巢及子宫的表达则说明,IGF-1能够调节卵泡和黄体的功能,从而影响卵巢中类固醇的生成;并参与了母胎界面的免疫耐受调节。同时IGF-1和IL-18的协同作用也为妊娠的维持创造了有利的免疫微环境。3. IGF-1和IL-18在妊娠各期大鼠的下丘脑变化主要在下丘脑视前室周核、视上核、视前大细胞核、视前内侧核、视前外侧核、视前交叉上核、弓状核等主要核团,说明下丘脑视前区的核团与妊娠调控密切相关。4.妊娠大鼠阴道壁注射不同剂量的IFN-γ后,下丘脑-垂体-性腺轴中IGF-1和IL-18的表达变化各不相同。注射100IU/g体重、500IU/g体重的IFN-γ后,下丘脑视前区视前室周核等核团中IGF-1和IL-18的表达均下调,下丘脑其它核团及垂体、子宫和卵巢中IGF-1和IL-18的表达也有不同程度的变化。在外周血中,妊娠早期的植入后期IGF-1和IL-18的表达水平有不同程度的下调。提示,高剂量的IFN-γ对妊娠期大鼠下丘脑-垂体-性腺轴中IGF-1和IL-18的表达及植入后期大鼠外周血中IGF-1和IL-18的水平均具有调控作用。表明IFN-γ对妊娠的调控是非常复杂和精细的。5.给妊娠早期大鼠注射高剂量的IFN-γ(500IU/g体重),会造成胚胎丢失和妊娠终止。这可能是IFN-γ不同程度的下调了子宫和卵巢中IGF-1和IL-18的表达及外周血中IGF-1和IL-18的水平而造成的妊娠终止,结果提示,注射高剂量IFN-γ是机体免疫调控紊乱,妊娠失败的原因之一。6.阴道壁注射小剂量(2IU/g体重)的IFN-γ,可上调流产大鼠子宫和卵巢中IL-18mRNA及IL-18蛋白表达的同时还可以上调外周血中IL-18的水平,使其接近正常妊娠组,结果提示外源注射小剂量的IFN-γ可以通过调节机体内一些细胞因子的水平,在维持妊娠中具有重要作用。7.米非司酮致大鼠流产后,其外周血血清中IGF-1和IL-18的水平均有所降低,而注射小剂量的IFN-γ其水平又有所回升,IFN-γ可能是通过调节妊娠大鼠下丘脑-垂体-性腺轴IL-18和IGF-1的水平来抑制流产引发的炎症过程,从而通过免疫途径调控并维持妊娠。提示临床检测妊娠早期大鼠外周血中IL-18和IGF-1的水平对于预防胚胎丢失和流产具有一定的参考价值。8. IFN-γ与IGF-1、IL-18的表达存在密切关系。正常妊娠时,注射高剂量IFN-γ引起IGF-1和IL-18的表达下调,打破了Th1/Th2型细胞因子平衡关系,造成妊娠终止。注射小剂量的IFN-γ则通过下丘脑-垂体-性腺轴的途径及血液反馈途径上调IGF-1和IL-18的表达,影响母胎界面的免疫微环境,调节流产大鼠Th1/Th2型细胞因子平衡,维持妊娠。IFN-γ在预防妊娠早期流产中发挥着重要作用。更多还原

【Abstract】 During pregnancy, the expression of several cytokines in the hypothalamus-pituitary gland-gonad axis were regulated through the complex neuro - endocrine - immune network, the changes or disorders of the expression of these cytokines caused by certain external factorsoften cause a variety of diseases and even pregnancy termination. Currently, the regulation and mechanism of the control and interaction of these cytokines during pregnancy were not confirmed. As reported that, Interleukin -18 (IL-18), insulin-like growth factor -1 (IGF-1) andγ-interferon (IFN-γ) are involved in the regulation of pregnancy, but the mutual regulation of these cytokines during pregnancy was less clear. In this research, immunohistochemical SP method was used to study the distribution of IGF-1 and IL-18 expression in the hypothalamus-pituitary gland-gonad axis during pregnancy, the influence between IFN-γ, IGF-1 and IL-18 expression, also image analysis system was use to analyzed its expression too. Fluorescent quantitative RT-PCR was used to detect the expression of IL-18 mRNA and IGF-1mRNA of the rat ovary and uterus after embryo implanted, and its expression after injection of IFN-γin the vaginal wall meanwhile, ELISA method was used to assay the expression of IL-18 and IGF-1 of the peripheral blood, and combined with microscopic and morphological analysis of the infection of IFN-γto pregnancy. All these offer the nerve biological research data for the mechanism of embryo loss during early pregnancy. The results of this research were as follows:1.During rat pregnancy, IGF-1 and IL-18 were expressed in a wide range of rat’s 21 thalamencephalon nuclei, such as hypothalamus, periventricular nucleus, supraoptic nucleus, preoptic large nucleus, medial preoptic nucleus, preoptic nucleus, suprachiasmatic nucleus, the bow shaped nucleus, and so on; existed in the adenohypophysis and the neurohypophysis; and also expressed in the ovarian granulosa luteal cells, the growth of follicles, mature follicles, endometrial stromal cells, uterine epithelial cells, uterine smooth muscle cells, and vascular endothelium. This showed that IGF-1 and IL-18 play an important way on the function of pregnancy through the pathway of hypothalamus - pituitary - gonadal axis.2.The expression of IGF-1 and IL-18 in the pregnant rat hypothalamus - pituitary - gonadal axis are different during the stages of pregnancy. The expression of IGF-1 in thalamencephalon was increased during early implantation, implantation, plant continues, lower during the middle of pregnant, and espression higher in the third trimester of pregnancy than the middle. The expressed of IL-18 in thalamencephalon was descended during early implantation and implantation, and significantly increased post-implantation. This showed that IGF-1 and IL-18 have different characteristics spatially and temporally. IGF-1 expression in embryo implantation and late pregnancy showed that IGF-1 had important role in regulating embryonic development and delivery, while expression in the ovary and uterus, showed that IGF-1 could regulate the function of follicle and corpus luteum, thus affecting the generation of ovarian steroid, and involved in the regulation of immune of the maternal-fetal interface. Therefore IGF-1 and IL-18 synergy created the immune microenvironment for the maintenance of pregnancy.3. IGF-1 and IL-18 expression in rat thalamencephalon was different during the pregnancy phase, mainly in the hypothalamus preoptic periventricular nucleus, supraoptic nucleus, preoptic large nucleus, medial preoptic nucleus, preoptic nucleus, suprachiasmatic nucleus, arcuate nucleus and other major nuclei, which indicated that the hypothalamic preoptic area was concerned with the regulation of the pregnancy. 4. The expression of IGF-1 and IL-18 of the hypothalamus - pituitary - gonadal axis in pregnant rats acted different after injecting with different doses of IFN-γ. In detail, after the IFN-γinjection of 100IU/g, 500IU/g to rats, the expression of IGF-1 and IL-18 in hypothalamic preoptic nucleus and other nucleus all decreased. The different expressions of IGF-1 and IL-18 were also observed in other nuclei of the hypothalamus and pituitary, uterus and ovary. In peripheral blood, The expressions of IGF-1 and IL-18 in the hypothalamus - pituitary - gonadal axis were decreased in a different extent. These results suggested that the high dose of IFN-γshowed the downregulation effecton the expression of IGF-1 and IL-18 in the pregnant rat hypothalamus - pituitary - gonadal axis and that in the peripheral blood during early pregnancy. This demonstrated that the regulation of IFN-γon the pregnancy was a complex process.5. High dose injection of IFN-γ(500 IU/g) to the early pregnant rats could result fetal loss and pregnancy termination. The possible reason was the downregulation of the IGF-1 in the uterine and ovarian and IL-18 in blood. This result suggested that high doses of IFN-γwas one of keys that disorder the disorder of immune regulation system.6. Low dose of IFN-γto aborted rats could increase the expression of IL-18mRNA and IL-18 protein, in the uterus and ovary. Meanwhile, it also increased the IL-18 level, which was close to the normal group. The result suggested that low dose of IFN-γcould regulate the level of some cytokines, which was important to the maintenance of pregnancy.7. After mifepristone induced abortion, the levels of IGF-1 and IL-18 in the serum were all decreased, however, a little re-increase has been determined after the injection of IFN-γ. The possible reason was the regulation of IL-18 and IGF-1 levels of the pregnant rats hypothalamus - pituitary - reproductive axis for the inhibition to the inflammation caused by abortion, also for the the maintenance of pregnancy. This result suggested that the levels of IL-18 and IGF-1 in the peripheral blood of early pregnancy rats, was something valuable to fetal loss and abortion.8. There was a close expression relationship among IFN-γand IGF-1, IL-18. During the normal pregnancy, the decrease of IGF-1 and IL-18 expression broke down the balance of Th1/Th2 type cytokines and resulted the termination of pregnancy. However, the low dose of IFN-γcould influence the immune microenvirionment of maternal-fetal interface, regulate the balance of Th1/Th2 type cytokines, and maintain pregnancy. This suggested that IFN-γact an an important role in the prevention of abortion in early pregnancy.更多还原