【作者】 徐静；

【导师】 李嫔；

【作者基本信息】 上海交通大学 ， 儿科学， 2016， 硕士

【摘要】 【目的】下丘脑-垂体-性腺轴的激活标志着青春发育的开始,该轴的过早启动与儿童中枢性性早熟的发生密切相关,然而其机制至今尚未阐明。2007年,利用高通量的基因芯片技术鉴定出大鼠和恒河猴下丘脑调控青春发育的基因网络,并得到愈来愈多研究结果的证实。目前认为青春发育并不是由单一的信号分子或信号通路所调控,而是由多种功能各异的基因所构成的网络协同作用的结果,其中EAP-1和CUX-1基因在这个网络中发挥着重要作用。本研究拟利用组织免疫荧光染色、实时荧光定量PCR以及Western blot技术以检测EAP-1和CUX-1在雌鼠青春发育前后的表达变化,探讨二者在下丘脑的表达分布及其与KISS-1、GnRH表达的关系。【方法】将雌性SD大鼠分为三组:幼年组(25日龄)、性发育早期组(35日龄)和性成熟组(42日龄)。通过实时荧光定量PCR技术测定不同发育阶段的雌性SD大鼠下丘脑KISS-1、GnRH、EAP-1和CUX-1mRNA水平,并进行弓状核(ARC)区域的组织免疫荧光检测,比较青春期前后EAP-1和CUX-1免疫反应阳性细胞的表达变化。采用Western blot的方法检测EAP-1蛋白在各个发育阶段的表达水平。【结果】1.下丘脑KISS-1 mRNA的表达水平在性发育早期组显著升高,与幼年组相比,差异具有统计学意义(P<0.01),性成熟组与之相比,差异无统计学意义(P>0.05)。2.GnRH mRNA的表达水平在性成熟组最高,与幼年组及性发育早期组相比差异均具有统计学意义(分别为P<0.01和P<0.05)。然而,EAP-1和CUX-1 mRNA的水平在各组间差异均无统计学意义(P>0.05),这与先前的研究结果相悖。3.组织免疫荧光染色显示,EAP-1和CUX-1蛋白均表达于神经细胞的胞核内,集中分布于以弓状核区域为主的第三脑室周围,部分CUX-1表达于Kisspeptin神经元。4.弓状核EAP-1阳性细胞数性发育早期组与幼年组相比显著升高,差异具有统计学意义(P<0.01),性成熟组与性发育早期组相比降低,差异亦具有统计学意义(P<0.05),性成熟组与幼年组相比差异无统计学意义(P>0.05)。EAP-1阳性细胞百分率性发育早期组与幼年组相比显著升高,差异具有统计学意义(P<0.01),性成熟组与幼年组相比差异亦具有统计学意义(P<0.05),性成熟组与性发育早期组相比差异无统计学意义(P>0.05)。5.CUX-1阳性细胞计数在各组间均无统计学意义(P>0.05)。6.下丘脑EAP-1蛋白的表达在性发育早期达峰值,与幼年期相比差异具有统计学意义(P<0.05),到性成熟期稍有下降,但性发育早期与性成熟期表达量比较,差异无统计学意义(P>0.05)。【结论】EAP-1在性发育早期表达增加,其表达趋势与KISS-1一致,并且在解剖结构上与kisspeptin神经元相邻,提示其可能通过调控Kisspeptin/GPR54系统而参与青春发育启动的过程。更多还原

【Abstract】 [Objective]The activation of the hypothalamus-pituitary-gonad axis represents the onset of puberty and is closely related with central precocious puberty of childhood.However,the neurobiological mechanisms are poorly understood.In 2007,using a combination of high throughput and bioinformatics strategies,a group of related genes including EAP-1 and CUX-1 were singled out from the hypothalamus of female nonhuman primates and rats.Increasing studies have demonstrated that no isolated gene or pathway is solely responsible for the pubertal regulation and that puberty is controlled by a gene network consisted of multiple functional genes.In this study,immunofluorescence staining,real-time PCR as well as western blotting were carried out to explore the expression and localization of EAP-1 and CUX-1,along with their relationship with KISS-1 and GnRH.[Method]Female SD rats were used in these studies.According to three different stages,juvenile(25 d of age),early puberty(35 d of age)and adult(42 d of age),animals were divided into 3 groups.10 rats in each group were killed,and the hypothalamus tissues were collected immediately for quantitative analysis of genes.The expression of EAP-1、CUX-1、KISS-1 and GnRN genes mRNA were measured with real-time PCR.β-actin was used as the internal standard.Rats of three groups with 6 in each group were killed,and brain tissues were collected for immunofluorescence staining.Medical imaging analysis software was used to count the numbers of EAP-1 or CUX-1 positive cells.The protein expression changes of EAP-1 were measured by western blotting.[Result]The KISS-1 mRNA level in the early-puberty group was significantly increased compared with the juvenile group(P<0.01),while the GnRH mRNA level reached a peak at the adult age(P<0.01 versus the juvenile group,P<0.05 versus the early-puberty group).However,EAP-1 and CUX-1 mRNA expression in the hypothalamus did not significantly changed during the different sexual development stages,as determined by real-time PCR(P>0.05),a finding inconsistent with previous results.Immunofluorescence staining results showed that EAP-1 and CUX-1 genes were localized to neuronal nuclei.Both were prominent in cells of the the arcuate nucleus(ARC)of the rat hypothalamus.Especially,CUX-1 gene was coexpressed in the Kisspeptin neurons.Moreover,the number and percentage of EAP-1 positive cells in the early-puberty group were both significantly more than the juvenile group(P<0.01),while the difference in the amounts of CUX-1 positive cells among the three groups was not statistically significant(P>0.05).The protein expression of EAP-1 in the early-puberty group was significantly higher than that in the juvenile group(P<0.05).[Conclusion]EAP1 expression increases at the early puberty in accordance with KISS1 gene and its localization is close to the kisspeptin neurons,which indicates that EAP-1 gene may be involved in the neuroendocrine control of female puberty in correlation with the Kisspeptin/GPR54 signal pathway.更多还原