【作者】 张艳；

【导师】 范光丽；

【作者基本信息】 西北农林科技大学 ， 基础兽医学， 2008， 博士

【摘要】 成功的胚胎植入以及随后的妊娠维持过程均要求细胞因子、激素和免疫因子在机体内正确分布和表达,一旦这些物质在时间和空间上的表达紊乱就会引发各种妊娠疾病,甚至造成流产。然而调控这一过程的许多生物化学信息分子表达的空间及时间特性还未完全阐明。近些年的研究发现,白细胞介素-18(Interleukin-18, IL-18)、诱导型一氧化氮合酶(Inducible nitric oxide synthase , iNOS)和核转录因子(Nuclear factor-kappa B,NF-κB)与生殖密切相关,但IL-18、iNOS和NF-κB在妊娠和流产中的作用及关系还不清楚。IL-18和iNOS可作为神经-免疫-内分泌的调节因子,因此,本试验采用超敏感的免疫组织化学SP法和图像分析法对非妊娠(发情期)、妊娠各期(胚胎植入前期、植入期、植入后期、妊娠中期和妊娠晚期)和早期流产大鼠下丘脑-垂体-性腺轴中IL-18和iNOS的分布及表达特点进行了研究,采用RT-PCR方法检测了非妊娠、正常妊娠早期及早期流产大鼠子宫中IL-18和iNOS mRNA的水平,用ELISA和硝酸酶还原法检测了外周血中IL-18和NO水平的变化。由于NF-κB是一种信号转导因子,主要在局部发挥作用,故本实验采用免疫组织化学SP法和图像分析法对非妊娠、妊娠各期和早期流产大鼠卵巢和子宫中NF-κB的分布及表达特点和激活程度进行了研究。主要结果如下: 1. IL-18和iNOS在非妊娠和妊娠各期大鼠下丘脑视前交叉上核、视上核、室旁核等24个核团均有表达,分布广泛;在腺垂体、垂体中间部和神经垂体纤维中也有分布;卵巢黄体颗粒细胞、卵泡颗粒细胞、卵泡膜细胞、卵母细胞、卵泡液、血管内皮细胞、间质腺、生殖上皮及基质中均存在;子宫腔上皮细胞、腺上皮细胞、基质细胞、子宫平滑肌细胞、血管内皮细胞及平滑肌细胞中也有表达。提示IL-18和iNOS可能以自分泌或旁分泌的方式参与下丘脑、垂体、卵巢及子宫功能的调节。2. IL-18和iNOS在下丘脑、垂体、卵巢及子宫中的表达量在妊娠的不同时期有所差异:在妊娠早期表达较多(除妊娠初期下丘脑IL-18水平较低),中期明显降低,而晚期又有所回升。提示IL-18和iNOS参与整个妊娠过程,并且在妊娠过程中的表达程度受到严格的时间和空间调控。从时间上看,IL-18和iNOS在胚胎植入前后和妊娠晚期表达较多,提示IL-18和iNOS在胚胎植入过程和分娩启动中发挥重要作用。从空间上看,IL-18和iNOS在下丘脑和垂体水平参与了对妊娠的调节,通过下丘脑-垂体-卵巢轴调控子宫母胎界面的微环境。妊娠过程中卵巢和子宫IL-18及iNOS的规律性表达表明,IL-18和iNOS可调节妊娠期卵泡和黄体功能,进而影响卵巢类固醇的生成;同时也参与了母体胎儿界面免疫耐受的调节。机体各部位的共同协作为胎儿的发育提供了有利的免疫微环境。3.在妊娠过程中大鼠下丘脑IL-18和iNOS水平的变化主要在视前区、视上区和漏斗部的弓状核,提示下丘脑的这些部位与生殖密切相关。而前连合核等个别核团表达状况特殊,提示他们可能还有其他生理功能。4. NF-κBp65在非妊娠和妊娠各期大鼠卵巢和子宫中均有表达,并呈规律性变化:在妊娠早期表达较多,中期明显降低,而晚期又有所回升,提示NF-κBp65参与妊娠过程中卵巢及子宫功能的调节,在胚胎植入和分娩启动中发挥重要作用。在整个妊娠过程中95%的NF-κBp65位于细胞质中,仅有5%位于细胞核,提示NF-κBp65活性处于抑制状态,有利于妊娠的维持。5.在妊娠早期孕鼠胎盘锥、妊娠中期和晚期胎盘中均有IL-18、iNOS和NF-κBp65表达,提示IL-18、iNOS和NF-κBp65可作为母体与胎儿间的信息传递因子,在胎盘和胎儿的发育过程中发挥重要作用。6.流产组大鼠外周血中IL-18和NO的水平明显低于同期正常妊娠组,下丘脑-垂体-性腺轴中表达的IL-18和iNOS也较正常妊娠和非妊娠组大鼠明显降低,提示下丘脑-垂体-卵巢IL-18和iNOS表达的减少可能是流产发生的原因之一。推测下丘脑-垂体-性腺轴中IL-18和iNOS表达减少,使得其对下丘脑、垂体和卵巢的内分泌功能的调节不足,各种激素和因子表达紊乱,最终使母胎界面的免疫微环境失衡,母体对胎儿产生排斥,诱发流产。7.流产时,大鼠下丘脑表达iNOS的小胶质细胞增多,而小胶质细胞的激活与炎症的发生密切相关,提示iNOS可能与小胶质细胞的激活有关,并在流产的炎症过程中发挥重要作用。8.与正常妊娠和非妊娠组大鼠相比,流产大鼠卵巢NF-κBp65的表达明显减少。NF-κBp65是凋亡抑制因子,推测NF-κBp65表达减少,不足以抑制凋亡基因的表达,导致卵泡和黄体颗粒细胞凋亡,卵泡闭锁,黄体溶解,诱发流产;流产大鼠子宫中NF-κBp65的表达明显增加,且32%NF-κBp65阳性产物位于细胞核内(正常妊娠仅为5%),提示子宫NF-κBp65的表达增加和过度激活可能是流产发生的原因之一。9.在妊娠和流产过程中大鼠下丘脑和垂体IL-18与iNOS水平的变化趋势基本一致,相关性分析表明二者呈正相关,提示二者在妊娠和流产过程中对下丘脑及垂体功能的调节具有协同作用。而卵巢中IL-18、iNOS和NF-кB的水平在妊娠和流产过程中变化趋势也一致,呈正相关,提示三者在妊娠和流产过程中对卵巢功能调节有协同作用。然而这三种活性物质在妊娠和流产过程中对子宫功能的调节存在差异:在妊娠过程中三者呈正相关,而在流产过程中IL-18与NF-кB呈正相关,iNOS与NF-кB呈负相关。综合这些物质在流产中的变化,推测在流产过程中NF-κB通过促进子宫肌IL-18 mRNA及蛋白的表达,抑制子宫内膜IL-18及子宫iNOS mRNA及蛋白的表达,使子宫的免疫调控失衡,诱发流产。更多还原

【Abstract】 Proper distribution and expression of cytokine, hormone and immune factor in organism is a prerequisite for successful implantation and subsequent pregnancy maintenance. If these factors distribute and express disorderly, pregnancy disease and abortion will initiate. But biochemical signals that control such events have not been well characterized. Recent research shows IL-18, iNOS and NF-кB hav close relation with reproduction. But their relation with pregnancy and abortion is unclear. IL-18 and iNOS may act as regulator factors among nerve, immunity and endocrine, so we studied the distribution and expression of IL-18 and iNOS in rat hypothalamus-pituitary gland-gonad axis during oestrus, all pregnancy period and early abortion by immunohistochemistry SP method, the expression of IL-18 and iNOS mRNA in rat uterus of oestrus, pregnancy 9 days and abortion in pregnancy 9 days by RT-PCR method, the level of IL-18 and NO in rat serum of oestrus, pregnancy 9 days and abortion in pregnancy 9 days. NF-κB may act as a signal transduction factor, so we studied the distribution and expression of NF-κB in rat ovary and uterus during oestrus, all pregnancy period and early abortion by immunohistochemistry SP method and oboserved the activation degree of NF-κB in all groups. The main result as follow:1. IL-18 and iNOS extensively distributed in 24 hypothalami nucleus of oestrus and pregnancy rat, such as pre-opticus suprachiasmatic nucleus, supraoptic nucleus, paraventricular nucleus. In rat pituitary, IL-18 and iNOS immunoactivity product were found in adenohypophysis cell, pituitary pars intermedia cell and neurohypophysis fiber; In rat ovary, IL-18 and iNOS mainly existed in ovarian follicular granular cell and theca cell, oocyte, follicular fluid, corepus luteam granular cell, vascular endothelial cell, interstitial gland, germinal epithelium and matrix. In rat uterus, IL-18 and iNOS mainly prensented in endometrial or decidual luminal epithelium, glandular epithelium, stroma or decidual cells, myometrium, vascular endothelial cell and vascular smooth muscle cell. The result shows IL-18 and iNOS may regulate the function of hypothalamus, pituitary gland, ovary and uterus in autocrine and paracrine ways.2. The expression of IL-18 and iNOS in rat hypothalamus-pituitary gland-gonad axis was different in different prgnancy period. The level of IL-18 and iNOS was higher in early pregnancy, lower in mid trimester of pregnancy and higher in late trimester of pregnancy. The result shows IL-18 and iNOS participate all pregnancy and their expression was controled strictly in time and space modes. In time mode, the level of IL-18 and iNOS was high in circa of embryo implantation and late trimester of pregnancy. It shows IL-18 and iNOS may have great roles in the process of embryo implantation and parturition. In space mode, IL-18 and iNOS in hypothalamus and pituitary may participate the regulation of all pregnancy process. They may control the function hypothalamus-pituitary gland-gonad axis to affect the microenveronment of mother-foetus interface. The expression of IL-18 and iNOS in ovary and uterus may regulate the function of ovarian follicle and corepus luteum, affect the production of ovary steroid and the immunotolerance in mother-foetus interface. These can protect foetus from the mother rejection and provide a good enviroment for foetus development.3. The level change of IL-18 and iNOS in hypothalamus mainly presented in preoptica area, supraoptic region and arcuate nucleus in fimbria. So it hints these hypothalamic parts may have close relation with reproduction. However, the level change tendency of IL-18 and iNOS in some nucleus, such as commissural nucleus, was unusual.It shows they may have other physiologic fuction.4. NF-κBp65 existed in rat ovary and uterus during oestrus and all pregnancy period. And the level NF-κBp65 presented a regulated change: higher in early pregnany, lower in mid trimester of pregnancy and increased in late trimester of pregnancy. The result shows NF-κBp65 may as a important regulator of the function of ovary and uterus and have great roles in the process of embryo implantation and parturition. NF-κBp65 in cytoplasm has incompentence.when it enters into nucleus, it has a activity. All pregnancy process, 95% NF-κBp65 main exist in cytoplasm and 5% in nucleus. The result shows NF-κBp65 in inhibition activity may promote the pregancy maintence.5. The expression of IL-18, iNOS and NF-κBp65 was found in rat ectoplacental cone in early pregnancy and placenta in mid trimester of pregnancy and late trimester of pregnancy. The result shows IL-18, iNOS and NF-κBp65 may act as information transfer factors between mother and foetus and play a great role in development placenta and foetus.6. Compare to the same norm pregnacy group, the level of IL-18 and NO in abortion rat serum and the expression of IL-18 and iNOS in abortion rat hypothalamus-pituitary gland-gonad axis were obviously decreased. The result shows the expression of IL-18 and iNOS in rat maybe a factor for inducing abortion. The decreased expression of IL-18 and iNOS in rat hypothalamus-pituitary gland-gonad axis may induce the regulation of IL-18 and iNOS on the endocrine function of hypothalamus- pituitary gland-gonad axis disorder, which could induced hormone and cytokine secreted disorderly and affected the microenveronment of mother-foetus interface. Finally, the balance of immunotolerance in mother-foetus interface was breaked, the rejection between mother and foetus increased, and abortion was induced.7. when rat aborted, the number of microglia which contain iNOS immunoactive product increased. Because of the activity of microglia have a close relation with inflammation development. So the result shows iNOS may activates microglia and play a important role in inflammation development during abortion process.8. Compare to the same norm pregnacy group and oestrus group, the expression of NF-κB p65 in abortion rat ovary was obviously decreased . NF-κBp65 is a anti-apoptosis factor. So we suppose the decreased expression of NF-κBp65 will not inhibit the expression of apoptosis gene and induce the ovarian follicular and corepus luteum granular cell apoptosis. Sequentia follicular atresia and luteolysis, the abortion is induced. However, the expression of NF-κBp65 in abortion rat uterus was notably increased and the cell number of NF-κBp65 in nucleus ( 32% compared with 5%)significantly increased. All results show the increased expression of NF-κBp65 and NF-κBp65 activity in rat uterus may as a factor for abortion genesis .9. In pregnancy and abortion process, the change tendency of IL-18 and iNOS in rat hypothalamus and pituitary gland is basic coincidence. By correlation analysis, it has positive correlation between IL-18 and iNOS. The result shows it has synergism between IL-18 and iNOS in regulating the function of hypothalamus and pituitary gland.In rat ovary, the change tendency of IL-18 , iNOS and NF-κBp65 in pregnancy and abortion process is basic coincidence too. The result shows their have synergism in regulating the function of ovary. However, in rat uterus the change tendency of IL-18, iNOS and NF-κBp65 in pregnancy process is different from that in abortion process. In pregnancy process, these factors have a positive correlation. It shows their have synergism in regulating the function of pregnancy uterus. In abortion process, it has a positive correlation between IL-18 and NF-κBp65, a negative between NF-κBp65 and iNOS. The result shows it has a synergism between IL-18 and NF-κBp65, antogonism between NF-κB p65 and iNOS, in regulating the function of uterus during abortion. Combined with the change of these substance in abortion process, it suppose NF-κBp65 is not only prompte the expression of IL-18 mRNA and protein in myometrium but also inhibit that in endometrial and the expression of iNOS mRNA and protein in uterus.更多还原