【作者】 初晨宇；

【导师】 高国栋； 黄威权；

【作者基本信息】 第四军医大学 ， 外科学， 2008， 博士

【摘要】 促性腺激素释放激素(GnRH)是由下丘脑神经元分泌的十肽小分子,促性腺激素释放激素受体(gonadotropin-releasing hormone receptor, GnRHR)属于七次跨膜的G蛋白耦联受体超家族。GnRH与GnRHR结合后,调节促性腺激素细胞分泌黄体生成素(luteining hormone ,LH)和卵泡刺激素( follicle-stimulating hormone ,FSH),在生殖过程中发挥重要作。促性腺激素包括FSH和LH,他们都是由α,β亚单位构成的二聚体糖蛋白。FSHR和LHR都是具有7个跨膜结构的G蛋白耦联受体。FSH和LH的分泌都受到下丘脑分泌的GnRH的调控。近年来的研究发现,GnRH及其受体还存在于下丘脑-垂体-性腺轴以外的脑组织、消化道、颌下腺、胰腺等消化器官以及前列腺癌等肿瘤细胞中,其功能也超出了生殖范畴。作为雌激素上游激素的GnRH及其受体在海马内也广泛存在,但未见有对其在缺血损伤下表达变化的报道。除垂体外,多种组织内均有FSH的存在。生殖系统中FSH可存在于前列腺,乳腺,睾丸,以及胎盘。在非生殖系统内可存在于胃的壁细胞,其功能也超出了生殖调控的范畴。FSHR除了在性腺中表达之外,也表达于输卵管上皮和子宫颈等部位。近年的研究发现,大鼠海马神经元中存在许多激素及其受体,其中包括作为生殖激素的GnRH、LH及它们的受体,但未见有存在FSH及其受体的报道。脑缺血再灌注损伤可以引发选择性的、延迟的神经元死亡,这种死亡主要是通过神经元的凋亡所引起的。近年来的研究已经表明,雌激素对海马CA1区缺血引起的神经元损伤和神经元凋亡具有保护作用,并且这种保护性作用是通过海马内的雌激素受体实现。GnRH及FSH是雌激素的上游激素,关于GnRH及FSH对海马神经元缺血再灌注损伤的保护性研究未见报道。由此我们进行了以下研究:应用原位杂交及免疫组织化学方法,结合图像分析系统,观察了缺血再灌注损伤后大鼠海马CA1区神经元GnRH及其受体的表达变化;应用原位杂交方法观察了FSH在大鼠海马的表达;应用免疫荧光组织化学双标记方法,在激光共聚焦显微镜下进行了大鼠海马FSH及其受体的共定位研究;应用免疫组织化学邻片双标记方法进行了大鼠海马FSH与GnRHR的共定位研究;通过制作大鼠MCAO模型、进行TUNEL染色的体内实验及制作大鼠海马神经元OGD模型、进行流式细胞仪Annexin V/PI双染色法检测的体外实验进行了FSH、GnRH对大鼠海马神经元缺血再灌注损伤的保护性研究。通过实验得到主要研究结果如下:大鼠海马CA1区神经元GnRH及GnRH mRNA的表达量随缺血再灌注损伤时间的延长而减少,阳性细胞数也随损伤时间延长而迅速减少。GnRHR及GnRHR mRNA的表达量随缺血再灌注损伤时间的延长先增加后减少,阳性细胞数随损伤时间延长而减少。损伤后3d, CA1区多型层及辐射层出现较多GnRH、GnRHR免疫反应及GnRH、GnRHR mRNA杂交信号弱阳性细胞;海马CA1至CA4区及齿状回的神经元呈FSH免疫反应阳性,同时也显示FSH mRNA阳性杂交信号,海马CA1至CA4区及齿状回的神经元呈FSHR免疫反应阳性,FSH和FSHR共定位于海马的同一神经元,FSH和GnRHR共定位于同一海马神经元;缺血再灌注损伤后72h,FSH干预组和GnRH干预组CA1区TUNEL染色阳性神经元平均灰度值大于对照组,即FSH干预组和GnRH干预组TUNEL染色阳性产物少于对照组。缺血再灌注损伤后72h,FSH干预组和GnRH干预组CA1区TUNEL染色阳性神经元数量比对照组少;FSH干预组、GnRH干预组与对照组相比,凋亡神经元百分比增加,存活神经元百分比减少。FSH干预组、GnRH干预组与无干预组相比,凋亡细胞百分比减少,存活细胞百分比增加。综上所述,本研究结果表明:缺血再灌注损伤后,大鼠海马CA1区神经元GnRH及GnRHR的表达发生变化,GnRH可能参与CA1区脑组织损伤后修复过程的调节;海马能表达FSH,海马的FSH神经元也是FSH作用的靶细胞。海马神经元产生的FSH可能通过旁分泌和自分泌途径对海马的功能进行调节,海马产生的GnRH也可能通过其受体对FSH神经元进行调节;预先给予一定浓度的FSH及GnRH干预,可以减少海马CA1区缺血再灌注损伤所引起的神经元凋亡。FSH及GnRH对神经元缺血再灌注损伤具有保护性作用。更多还原

【Abstract】 The gonadotropin-releasing hormone (GnRH), also called luteinizing hormone-releasing hormone (LHRH), is a decapeptide. Gonadotropin-releasing hormone receptor (GnRH receptor) is a member of the seven-transmembrane, G-protein-coupled receptor family. GnRH combined with GnRH receptor to regulate secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and plays an important role in reproductive process.Gonadotropine comprise of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). They are heterodimer glycoproteins composed ofαandβsubunits and have important roles in production of gonadal hormone and regulation of reproductive process. FSH receptor and LH receptor are G protein-coupled receptors. The secretion of FSH and LH are all regulated by GnRH secreted from hypothalamus. Increasing evidence suggests that GnRH and its receptor can also exist in other extrahypothalamic regions, such as gastrointestinal tract, submaxillary gland, pancreas and some malignant tumor cells such as prostate cancer cells. At the same time, the function of GnRH is beyond reproduction. GnRH, an upstream hormone of estrogen, which can regulate the secretion of estrogen, also broadly exists in hippocampus with its receptor; but there is no report about the change of expression of GnRH and its receptor in hippocampus with ischemia-reperfusion injury. Many research showed that FSH also exists in other tissues besides pituitary. In reproductive system, FSH exists in prostate, breast, testis and placenta; in non-reproductive system, FSH exists in parietal cells of stomach. Its functions are also beyond reproduction. Besides expressed in gonad, FSH receptor also exists in uterine tubal epithelium and cervix.Recently, research has showed that many hormones and their receptors, including reproductive hormones such as GnRH,LH and their receptors, exist in hippocampal neurons. However, whether FSH and its receptor also exist in hippocampal neurons remained unknown yet. Global ischemia-reperfusion injury elicits selective, delayed neuronal death, and the main reason is apoptosis. Many researches have showed that estrogen can protect neuron injury and neuron apoptosis in hippocampal CA1 region during ischemia and this protection is mediated by estrogen receptors in hippocampus. GnRH and FSH are upstream hormones of estrogen, but there is no report about their protection of ischemia-reperfusion injury in rat hippocampus.On the base of our previous research, we did the following experiments: combined with image analysis system, Immunohistochemistry and in situ hybridization method were used to observe the change of expression of GnRH and its receptor in neurons of CA1 region in rat hippocampus after ischemia-reperfusion injury; in situ hybridization method was used to observe the expression of FSH in rat hippocampus; double label immunofluorescence histochemical staining and confocal laser-scanning microscope were used to study the colocalization of FSH and FSHR in rat hippocampus; in vivo study of middle cerebral artery occlusion model building and TUNEL staining was made and in vitro study of oxygen-glucose deprivation model building and double staining of Annexin V/PI with flow cytometer was made to study the protection of ischemia-reperfusion injury by FSH and GnRH in rat hippocampus.Through these experiments, the results are as follows: the expression of GnRH and GnRH mRNA in CA1 reagion of rat hippocampus decreased with time after ischemia-reperfusion injury. the number of positive cells also decreased with time after injury. The expression of GnRHR and GnRHR mRNA in CA1 reagion of rat hippocampus increased at first and then decreased at last with time after ischemia-reperfusion injury. The number of positive cells decreased with time after injury. Three days after ischemia-reperfusion injury, not only GnRHR positive cells but also GnRH positive cells appeared at stratum oriens and stratum radiatum. Hippocampal neurons from CA1 to CA4 region and dentate gyrus showed both FSH immunoreactivity and FSH mRNA hybridization signal. Hippocampal neurons from CA1 to CA4 region and dentate gyrus showed FSHR immunoreactivity. FSH and FSHR co-located in the same hippocampal neurons. FSH and GnRHR co-located in the same hippocampal neurons. 72h after ischemia-reperfusion injury, mean grey levels of TUNEL stain positive neuron in CA1 region of rat hippocampus in FSH interfere group and GnRH interfere group were higher than that of control group, namely the TUNEL positive product in FSH interfere group and GnRH interfere group were less than that of control group.The number of TUNEL stain positive neuron in FSH interfere group and GnRH interfere group were less than that of control group. Compared with that of control group, the percent of apoptosis neuron in FSH interfere group and GnRH interfere group were increased while the percent of survival neuron decreased. Compared with that of no interfere group, the percent of apoptosis neuron in FSH interfere group and GnRH interfere group were decreased while the percent of survival neuron increased.In conclusion, our research found that the expression of GnRH and GnRHR in CA1 region of rat hippocampus make change after ischemia-reperfusion injury.GnRH might participate in the regulation of brain repair process at CA1 region after injury. Rat hippocampus can express FSH. FSH positive hippocampal neurons were also the FSH target cells. FSH secreted by hippocampal neurons might regulate the function of hippocampus through paracrine or autocrine. GnRH secreted by hippocampal neurons might regulate the FSH positive hippocampal neurons through GnRH receptor. Interfere with certain concentration of FSH or GnRH before ischemia-reperfusion injury can reduce the neuron apoptosis at CA1 region of rat hippocampus.FSH and GnRH have protective effect on neuron ischemia-reperfusion injury.更多还原