摘要
神经系统是机体最重要和最复杂的系统,它谜一样的发生过程一直吸引着发育神经生物学家的关注。而其中神经管的发生是关系到建立中枢神经系统(Central nervous system, CNS)原基的重要胚胎学事件,在这个过程中,神经板必须准时准确地关闭形成神经管,神经系统才能得以正常发育,否则将出现神经管缺陷(neural tube defect,NTD)和随之而来的脊柱裂或无脑等常见畸形。NTD的发病率在世界范围内占出生婴儿的0.1~0.9%,给家庭和社会带来了严重负担。但是迄今对此复杂过程的基因表达与确切调控途径均知之甚少,所以正常神经管形成的分子机制和NTD的发病机理已成为当今神经科学研究的热点和前沿。研究发现,胚胎9.5天(E9.5d)时,作为转录因子的信号转导和转录激活因子-3(signal transducer and activator of transcription factor-3,STAT3)在早期胚胎发育中的脑区有较高水平的表达,而E9.5d正是神经管关闭的重要时期,这是否提示STAT3有可能参与神经管的关闭呢?它的表达异常是否和NTD的发生相关呢?
神经管闭合后,神经管上皮细胞的进一步增生、分化和迁移,它的前端呈现三个膨大,依次称为前脑泡、中脑泡和菱脑泡,前脑泡发育为端脑和间脑,中脑泡发育为中脑,菱脑泡的前部发育为后脑,以后演变为脑桥和小脑,菱脑泡的后部发育延髓,神经管尾端形成未来的脊髓。神经管上皮细胞的增生、分化和迁移对于神经系统正常结构和功能的建立十分重要。近年研究发现,有许多基因参与中枢神经系统发育的调控,外环境也是通过这些基因发挥作用的。作为转录因子的STAT3参与很多基因的表达和调控,并与其他转录因子形成复杂的调控网络。在胚胎神经系统尤其是神经上皮内检测到STAT3的表达,它是否可能通过启动不同靶基因表达来参与神经管上皮细胞的增生、分化和迁移呢?
研究提示,成纤维细胞生长因子(fibroblast growth factor, FGF)参与调节早期神经发育阶段的许多关键过程,对鸡胚的研究发现FGF可能作为直接的神经诱导分子,其中FGF-8只在胚胎的早期增殖和神经发生阶段表达,涉及神经的早期发育和分化。已知FGFs是激活STAT的重要生长因子之一,并且FGF2与受体结合后可以激活STAT3,
由于FGF家族成员之间序列有高度的同源性,提示FGF8也可能激活STAT3而启动基因转录。FGF8是否可能作为STAT3上游的分子和STAT3共同促使神经管关闭呢?迄今对神经系统发育尤其是神经管关闭和NTD复杂过程的基因表达与调控的研究刚刚起步,涉及此过程的基因与具体调控途径均知之甚少,目前对正常神经管形成和NTD发生过程中的信号转导通路及分子机制的研究也尚无明确定论。
本实验采用全胚胎原位杂交技术、免疫组织化学技术、免疫细胞荧光染色技术等方法,观察到了STAT3在胚胎发育中时空表达规律,尤其在神经胚形成阶段和神经上皮增生、分化和迁移中的表达变化规律;并利用喂服过量维甲酸(retinoic acid, RA)诱导NTD模型,观察发生NTD的胚胎STAT3的表达变化;同时采用全胚胎原位杂交技术观察FGF8在正常胚胎神经胚形成(neurulation)过程中的表达和在NTD中的表达变化,并比较了FGF8和STAT3变化规律及其关系,旨在探讨STAT3在正常神经上皮发育及神经管关闭过程中的作用,试图揭示STAT3信号转导通路在胚胎神经系统发育过程中的作用和可能的分子机制。结果如下:
一、STAT3在小鼠胚胎神经系统发育中的表达
1、采用全胚胎原位杂交技术可观察到E8.75~11.5d小鼠胚胎STAT3的表达情况,在E8.75d时整个鼠胚几乎没有观察到STAT3 mRNA的表达,在E9.5d,随着神经管的逐渐关闭,STAT3 mRNA在前脑泡急剧增多,到E10.5d,阳性信号由前脑泡转移至中脑泡,E11.5d时仅在后脑泡的一狭窄区域检测到STAT3 mRNA的表达。STAT3胚胎神经组织的表达,尤其是神经管头段STAT3的表达及其时空变化,提示STAT3有可能参与了这个时期神经管的关闭以及神经管头段的发育。
2、本实验用酶免疫组织化学方法观察到在E13.5d,在侧脑室周围即未来大脑新皮层区域、间脑、发育中的视网膜、第四脑室周围的脑组织(未来的脑桥)、脊髓、三叉神经节和背根神经节C1均可观察到较强的STAT3阳性信号。E13d正是神经上皮套层细胞开始向边缘层迁移的时候,此时各脑区大量细胞表达STAT3,提示STAT3可能参与了神经前体细胞的分化和迁移。用荧光免疫组织化学方法观察,E14.5d和E15.5d时,大脑皮层、间脑、视网膜、第四脑室周围以及发育中的脑桥和脊髓等部位均可观察到发中等强度荧光的细胞,提示在细胞基本完成迁移和分化后,STAT3的表达有所下降。
二、STAT3与RA致小鼠NTD的关系
1、与正常对照组相比,喂服RA后孕鼠在E9.5~10.5d可观察到前脑顶部和后脑未闭合的畸形胚胎,在少数胚胎可观察到整个神经管未闭合,呈开放状态。表明该剂量的RA可以造成CNS发育的缺陷。
2、采用全胚胎原位杂交技术可以观察到RA诱导所致NTD畸形的鼠胚STAT3表达变化的情况,在E9.5、10.5d时 STAT3 mRNA的表达较正常胚胎减少,在前脑泡和中脑泡未观察到明显的STAT3 mRNA表达;E11.5d的胚胎神经管已关闭,与正常鼠胚相比STAT3 mRNA的表达无明显变化。研究发现,STAT3分子可以启动一些促进细胞存活的基因转录,如Bcl-2和Bcl-XL,从而维持细胞的存活。NTD发生时伴有的STAT3表达下调,这可?
The development of neural tube is an important embryologic event involved in the primordium establishment of the central nervous system. During this process, nervous system can’t develop normally until the neural plate closes on time accurately, or the neural tube defect (NTD) would occur. Nowadays, the studies of the developmental neurobiology have focused on the molecular mechanisms of the development of nervous system. Some studies have revealed that signal transducer and activator of transcription factor-3 (STAT3) was expressed in the developing brain area strongly at embryo 9.5d (E9.5d), when neural tube closed. Could it be possibe that STAT3 may be involved in the closure of neural tube and its changes were related with NTD?
Following the closure, neuroepithelium continued to prolife, differentiate and migrate, and prosencephalon, mesencephalons and rhombencephalon appeared in the anterior region of neural tube. The proliferation, differentiation and migration of the neuroepithelium are essential for the structural formation and functional establishment of the nervous system. Recent studies have revealed that a number of genes participated in the regulation and control of development of the central nervous system and some environmental factors exert their functions via these genes as well. As a transcription factor, STAT3 participated in the control of their target genes’ expression and forms a complicated network together with other transcription factors. Could the expression of STAT3 protein in the neuroepithelium suggest that STAT3 may contribute to the proliferation, differentiation and migration of neuroepithelium by promoting the expression of its target genes?
Fibroblast growth factors (FGFs) have recently been implicated as regulators of many key processes during early neural development. Studies in avian embryos have shown that FGF can induce the development of neural tissue. FGF8 has been shown to be expressed only during the early embryonic proliferation and neurogenesis, suggested a role it played in the early neural development and differentiation. It is known that FGF is one of the
growth factors which can activate STATs. FGF2 can activate STAT3, which suggested that FGF8 could activate STAT3. Could both of them be involved in the closure of neural tube? However, at present the studies about the gene expression, regulation and control of the complicated process of the nervous system’s development have just started, especially those relating to neural tube closure and neural tube defect (NTD). And very little is known about the correlative genes and relevant pathways.
In the present study, the expression and changes of STAT3 mRNA and protein were observed during the embryonic development using the whole-mount embryo in situ hybridization, immunohistochemistry etc; the changes of STAT3 mRNA expression were also observed in NTD by establishing a model of retinoic acid (RA)-induced NTD through adminisitrating overdose RA; the expression of FGF8 mRNA during neurulation and expression changes in NTD were also examined. Finally, a comparison of the expression and ralationship between the expression changes of FGF8 mRNA and STAT3 mRNA were carried out in order to investigate the roles of STAT3 in the development of neuroepithelium and the closure of neural tube, trying to reveal the function of this pathway and possible molecular mechanism in the developing nervous system. The results are as follows:
Ⅰ.The expression of STAT3 in the developing nervous system of mouse
1. The expression of STAT3 mRNA was detected from E8.75d to E11.5d by whole-mount embryo in situ hybridization staining. At E8.75d, STAT3 mRNA was hardly detected in the embryo; at E9.5d, STAT3 mRNA increased in the prosencephalon dramatically with the closure of neural tube; at E10.5d, STAT3 mRNA was observed in the mesencephalon; at E11.5d, STAT3 mRNA was limited to a narrow region in the metencephalon. The expression of STAT3 mRNA in the embryonic nerve tissue, especially the expression and spatio-temporal changes of STAT3
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