【作者】 苏娟；

【导师】 雷治海；

【作者基本信息】 南京农业大学 ， 基础兽医， 2008， 博士

【摘要】 Orexin是近年发现的一种神经肽,分为orexin A和orexin B。Orexin通过其受体发挥作用,参与摄食、能量稳态、睡眠和觉醒周期、生殖、内脏活动和神经内分泌等多种生理功能的调节。有关orexin参与生殖调控的资料大多数是在鼠上获得的,并且相关研究较少。关于orexin在猪体内的分布定位及其参与猪生殖调控的研究未见报道。因此,本实验以苏种猪为研究对象,采用免疫组织学、荧光双标记并结合激光共聚焦显微镜技术、real time-PCR和放射免疫测定(RIA)等方法研究了orexin B在猪体内的分布定位,orexin B在前脑与GnRH和OB-Rb的形态学关系,侧脑室注射orexin A后对下丘脑和垂体中相关神经肽的表达和血清PRL、FSH、Leptin及insulin水平的影响以及体外orexin对下丘脑脑片、腺垂体和卵巢颗粒细胞GnRH、LH和E2释放的影响,以期为orexin调控猪的生殖活动提供形态学和生理学资料。一、Orexin B在猪体内的分布定位本实验用免疫组织化学方法研究了10头1～2月龄苏钟猪脑、脊髓、部分内脏器官和内分泌腺内)orexin B免疫阳性细胞的分布定位及其纤维的投射,结果如下:1 Orexin B免疫阳性细胞在猪前脑、脑干、脊髓、部分内脏器官和内分泌腺均有分布。在前脑orexin B免疫阳性神经元胞体主要分布于视前区、下丘脑前区、室旁核、室周核、后区、背内侧核、外侧区、穹隆周核、未定带、乳头体核等部位。在脑干,orexin B免疫阳性神经元分布于从中脑到延髓的许多核团,包括前庭神经核、蓝斑、孤束核、下橄榄核和中缝苍白核等。在脊髓,orexin B免疫阳性神经元胞体,分布于颈部脊髓背角。在内脏器官,orexin B免疫阳性细胞主要分布于卵巢、睾丸、附睾、胃、十二指肠、空肠、回肠、结肠、胰和脾脏;在内分泌腺,主要分布于垂体和甲状腺。2 Orexin B免疫阳性纤维在中枢神经系统都有广泛的分布。在端脑,orexinB免疫阳性纤维在大脑皮质、隔区、终纹床核、杏仁核和斜角带均有分布。大脑皮质内也有少量阳性纤维分布。在间脑,主要分布于缰核、背内侧核、室周核、视上核、视交叉上核和穹隆周核,在乳头体上核、腹内侧核、下丘脑前区、后区和弓状核/正中隆起也有中等量分布。在脑干,前丘、后丘、脚间核、中缝背核和中央灰质等有密集的阳性纤维分布。在脑桥,蓝斑,被盖背侧核有大量的阳性纤维分布,在第四脑室底、极后区、臂旁内侧核有中等量的阳性纤维分布。此外,在小脑、脊髓和垂体柄及垂体后叶也有少量的阳性纤维的分布。二、在猪前脑orexin B与GnRH和OB-Rb的形态学关系本实验采用荧光双标记并结合激光共聚焦显微镜技术分析了10头1～2月龄苏钟猪前脑内orexin B与GnRH和OB-Rb之间的形态学关系。研究结果显示:在前脑的隔区、终纹床核和整个下丘脑(包括视前区、结节区和乳头体区),均见部分orexin B和GnRH在轴突中的共存;在视前区吻侧部、视前外侧区,GnRH神经元胞体较大,orexin B阳性纤维投射到GnRH神经元的胞体上;在下丘脑前区、视上核和视交叉上核存在少量的orexin B和GnRH在同一神经元中的共存。从前脑的隔区到正乳头体区,大约有30%左右的GnRH神经元胞体接受orexinB免疫阳性纤维的投射,其中有6%的是GnRH和orexin B在同一神经元中共存。在下丘脑前区、室周核、室旁核、未定带、外侧区和腹内侧核发现orexin B神经元内有OB-Rb免疫阳性反应。三、去卵巢猪侧脑室微量注射orexin A对下丘脑和垂体中相关基因表达的影响为探讨orexin A对生殖调控的途径。本实验采用real time PCR方法,研究了10头去卵巢苏钟母猪侧脑室注射orexin A或生理盐水后,对下丘脑PRL、FSH、leptin及OB-Rb mRNA表达的影响,结果如下:1侧脑室注射orexin A后,垂体内PRL mRNA的表达显著降低。2侧脑室注射orexin A后,leptin及OB-Rb mRNA的表达显著降低,而OX1R mRNA的表达显著升高,OX2R mRNA有升高的趋势但差异不显著。四、去卵巢猪侧脑室微量注射orexin A对血清PRL、FSH、leptin及insulin水平的影响Orexin是摄食、能量平衡和生殖之间的信号分子,在动物的营养状态和生殖之间起着桥梁作用。为此,我们研究了向去卵巢猪侧脑室注射orexin A对血清PRL、FSH、leptin及insulin水平的影响。结果如下:1侧脑室注射orexin A后,可引起血清中leptin水平显著下降。注射orexinA 10 min后开始下降,在30 min时降至最低,与对照组相比差异极显著(P＜0.01),随后,有所上升,但与对照组相比差异显著。其作用可持续80 min(P＜0.05)2侧脑室注射orexin A后,可引起血液中insulin显著升高。注射orexin A10 min后开始上升,在30 min时升至最高,与对照组相比差异极显著(P＜0.01),随后,有所下降,但与对照组相比差异显著。其作用可持续110 min(P＜0.05)3侧脑室注射orexin A能够抑制血清中PRL的水平。注射10min开始降低,注射后第40min时降到最低,与对照组相比差异极显著(P＜0.01)。其抑制作用可持续80 min(P＜0.05)4侧脑室注射orexin A后,血清中FSH有所上升,但差异不显著。五、Orexin对性成熟青年母猪下丘脑组织-垂体-卵巢轴作用的体外研究为探讨orexin在生殖调节中直接作用的靶位点,我们在体外培养了猪下丘脑脑片、垂体和卵巢细胞,研究了orexin A和orexin B在体外对下丘脑、腺垂体和卵巢GnRH、LH和E2释放的影响。结果显示:1 Orexin A(100和1000 nM)能显著的刺激下丘脑脑片GnRH的释放,差异极显著(P＜0.01),而orexin B只有1000 nM剂量组能显著刺激GnRH的释放,差异极显著(P＜0.05)。2 Orexin A(10、100和1000 nM)均能剂量依赖性地刺激腺垂体细胞LH的释放(P＜0.05),其中100和1000 nM剂量组与对照组相比差异极显著(P＜0.01)。orexin B(100、1000 nM)也能极显著刺激腺垂体细胞LH的释放(P＜0.01)。3 Orexin A(10、100和1000 nM)均能显著刺激卵巢颗粒细胞E2的释放,100nM剂量对卵巢颗粒细胞E2的释放的刺激作用最强,1000nM剂量组的刺激作用反而比100nM剂量组相比的有所下降。Orexin B 100和1000nM剂量组能显著的刺激卵巢颗粒细胞E2的释放(p＜0.05),其中1000 nM剂量组与对照组相比,差异极显著(p＜0.01)。上述研究表明,orexin B在猪下丘脑、垂体和卵巢均有分布,为orexin参与生殖调控提供了形态学依据。Orexin B在猪前脑与GnRH和OB-Rb有共存,提示Orexin参与生殖活动的调节可能是直接作用于GnRH神经元发挥作用也可能是经由leptin介导。侧脑室注射orexin A后,orexin能抑制下丘脑和垂体中PRL、leptin及OB-Rb mRNA的表达,可促进OX1R mRNA的表达,并降低血清中PRL、leptin和升高血清胰岛素的水平。体外试验研究表明orexin A和orexin B能够刺激下丘脑脑片GnRH、腺垂体和卵巢颗粒细胞LH和E2的释放。这些结果表明orexin既可直接影响生殖相关激素的分泌和释放,又可能通过leptin及胰岛素等摄食相关激素间接参与生殖活动的调节。本研究为orexin参与生殖调控提供了形态学和生理学资料。更多还原

【Abstract】 Orexin,a recently discovered neuropeptide,are divided as orexin A and orexin B. Orexin roles are mediated by it’s two different subtypes receptor(OXR)(OX1R and OX2R).Orexin has been implicated in the regulation of food intake,arousal states, energy metabolism,activity of visceral organs,neuroendocrine and reproduction.The datas that orexin involved in the regulation of reproduction were got mostly from rat and sheep,and very limited.Moreover,the distributions of orexin B and regulation of reproduction by orexin in swine were not reported in present literatures.Therefore, the distribution of orexin B in Suzhong swine,the relationship with GnRH and OB-Rb in the forebrain,expression of reproduction related neuropeptide and hormone mRNA in the hypothalamus and pituitary and PRL,FSH,leptin and insulin levels in serum after orexin A injected into lateral ventricle in ovariectomized swine,and effect of orexin on hypothalamus-pituitary-ovary in vitro were studied in present study with immunohistochemistry,double-labelled immunofluorescence combined laser-scanning confocal microscopy,real time-PCR and radioimmunoassay(RIA),in order to provide the morphological and physiological data for orexin role in regulating reproduction of swine.1 Localizatin of orexin B immunoreactive cells in swine10 Suzhong swines,1-2 months old,were used in this study in order to investigate localization of orxin B in the brain,spinal cord,some visceral organs and endocrine glands of swine with immunohistochemical method.The results were as follows:(1) Orexin B immunoreactive neurons were observed in the forebrain,brainstem, spinal cord,some visceral organs and endocrine glands in swine.In the forebrain, orexin B immunoreactive neurons mainly localized in the preoptic area,anterior hypothalamic area,periventricular and paraventricular nuclei,posterior hypothalamic area,dorsomedial hypothalamic nucleus,lateral hypothalamic area, perifornical area,zona incerta,mammillary area and so on.In the brainstem,orexin B immunoreactive neurons distributed in many nuclei,including the vestibular nuclus, locus coeruleus,solitary tract nucleus,inferior olivary nucleus and raphe pallidus.In the spinal cord,orexin B immunoreactive neurons only presented in the dorsal horn. In visceral organs,orexin B immunoreactive cell were observed mostly in the ovary, testicle,epididymis,stomach,duodenum,jejunum,ileum,colon,pancreas and spleen. In endocrine glands,orexin B immunoreactive cells were distributed in the pituitary and thyroid gland.(2) Orexin B immunoreactive fibers projected extensively in the the central nervous system.In the telencephalon,orexin B immunoreactive fibers projected to the cortex, septal area,bed nucleus of stria terminalis,caudate nucleus,amygdaloid nucleus and diagonal band.In the diencephalons,orexin B immunoreactive fibers densely projected to the habenular nucleus,dorsomedial hypothalamic nucleus,periventricular nucleus,supraoptic and suprachiasmatic nuclei.There were moderately dense orexin B immunoreactive fibers in the supramammillary nucleus,ventromedial nucleus, anterior hypothalamic area,posterior hypothalamic area,arcuate nucleus/median eminence.In the brain stem,there were very dense orexin B immunoreactive fibers in the superior colliculus,inferior colliculus,interpeduncular nucleus,dorsal raphe nucleus and central gray.Abundant orexin B immunoreactive fibers also projected to the pontine nucleus,locus coeruleus and the dorsal tegmental nucleus.Moderate numbers of orexn B fibers were observed in,area postrema,fourth ventricle and parabrachial nucleus.In addition,Small numbers of orexn B fibers were observed in the cerebellum,pituitary and spinal cord.2 The relationship of orexin B with GnRH and OB-Rb in the pig forebrain10 Suzhong swines,1-2 months old were used in this study in order to investigate the morphological relationship between orexin B and GnRH,orexin B and OB-Rb using double-labelled immunofluorescence combined laser-scanning confocal microscopy.The results showed that approximately 30%of GnRH fibres and neurons were contacted by orexin B fibres from the septal area and OVLT to the arcuate nucleus. Approximately 6%GnRH perikarya overlapped with orexin B perikarya The overlapped neurons were mainly concentrated in the supraoptic nucleus.In the anterior hypothalamic area,periventricular,paraventricular nucleus,zona incerta, lateral hypothalamic area and ventromedial nucleus,the orexin B neurons expressed OB-Rb. 3.The effect of orexin A on expression of reproduction related neuropeptide and hormone mRNA in the hypothalamus and pituitary after injection of orexin A into the lateral ventricle in ovariectomized swineThe expression of PRL,leptin,OB-Rb and OXRS mRNA in the hypothalamus and pituitary was studied with real time PCR in 10 ovariectomized sows after injection of orexin A or physiological saline into the lateral ventricle in order to approach the pathway through which orexin A regulate pig reproduction.The results were as follows:(1) After injection of orexin A into the lateral ventricle,it depressed expression of PRL mRNA in the pituitary.(2) After injection of it into the lateral ventricle,orexin A depressed expression of leptin,OB-Rb mRNA,and promoted expression of OX1R mRNA in the hypothalamus and pituitary.4 The effect of orexin A on serum PRL、FSH、leptin and insulin level after injection of orexin A into the lateral ventricle in ovariectomized swineHormone levels in serum was analyzed with radioimmunoassay(RIA) from 1.5 h before injection to 3 h after injection of orexin A or physiological saline into the lateral ventricle in 10 ovariectomized swines in order to study the effect of orexin A on PRL,FSH,leptin and insulin secretion(blood sample interval was 10 min).The results were as follows:(1) Orexin A depressed the secretion and release of leptin after injection of orexin A into the lateral ventricle,and leptin concentration in serum began to descend at 10 min,was down to the lowest point at 30 min after injection,and maintained the low level for 80 min.(2) Orexin A promoted the secretion and release of insulin after injection of orexin A into the lateral ventricle.Insulin concentration in serum began to increase 10 min, was up to the peak 30 min after,and returned to the initial level at 110 min after injection.(3) Orexin A depressed the secretion and release of PRL after injection of orexin A into the lateral ventricle.PRL concentration in serum began to descend at 10 min, was down to the lowest 40 min after injection,maintained the low level for 80 min.(4) Orexin A had no significant influence on FSH level in plasm after injection of orexin A into the lateral ventricle.5.The effect of orexin on the hypothalamus-pituitary-ovary axis in vitro To study the action target of orexin,we studied hypothalamic explants anterior pituitary and ovary gransola cells in vitro.Our results were as follows: (1) After hypothalamic explants were challenged for 45 min with orexin A(1,10,100 and 1000 nm),the GnRH levels in the incubated aCSF were measured by radioimmunoassay(RIA).Orexin A(100 and 1000 nM) stimulated the release of GnRH from hypothalamic explants harvested from prepuberal swine.However,only 1000 nM orexin B could stimulated the release of GnRH from the hypothalamic explants harvested from prepuberal swine.(2) orexin A(10,100 and 1000 nm) could dose-dependent stimulate the release of LH from anterior pituitary cells.And orexin B(100,1000 nM) also could stimulate the release of LH from anterior pituitary cells.(3) Orexin A(10,100 and 1000 nM) could stimulate the release of E2 from ovary gransola cells.But,the 100 nM dose was more effective than 1000 nM.Orexin B(100 and 1000 nM) also could stimulate the release of E2 from ovary gransola cells.The above studies showed that orexin B was observed in the hypothalamus, pituitary and ovary of swine,which provided the morphologic data for orexin role involving in regulation of pig reproduction.Orexin B colocalized with GnRH and OB-Rb suggested that orexin could directly act to GnRH neuron or indirectly act to leptin participate in regulating reproduction.After injection of orexin A into the lateral ventricle,it depressed the expression of PRL,leptin and OB-Rb mRNA and promoted the expression of OX1R mRNA in the hypothalamus and pituitary, Meanwhile,it depressed PRL,leptin and increased insulin level in serum,and orexin could stimulate the release of GnRH,LH and E2 from hypothalamus plants,the anterior lobe of pituitary and ovary gransola cells.This investigation suggested that orexin be involved in regulation of reproduction either by stimulating directly release of GnRH and LH,or via affecting indirectly release of GnRH and LH by the way of leptin and insulin.These studies provided morphological and physiological data for orexin role in regulating reproductive activity.更多还原