【作者】 李红丽；

【导师】 蔡文琴；

【作者基本信息】 第三军医大学 ， 人体解剖与组织胚胎学， 2003， 博士

【摘要】 周期素依赖性蛋白激酶5(cyclin-dependent kinase，CDK5)作为CDKs家族的成员之一，是上世纪90年代初才发现的一个多功能的蛋白质。研究证实CDK5主要通过对不同的信号传导通路上靶蛋白的磷酸化来参与脑发育早期神经细胞的迁移、轴突生长等重要发育事件的调节。神经胚形成(neurulation)是神经系统早期发育的重要事件，其组织、细胞、特别是分子机制的研究是当今发育神经生物学的一个热点领域。近年来人们认识到胚胎早期神经管的神经上皮中存在着大量能够增殖分化的多潜能的神经前体细胞即神经干细胞。从基因水平而言，神经管关闭的过程是一系列基因按高度特异的时空模式表达与相互作用的结果；众多遗传或环境因素的改变均可导致包括神经管缺陷(neural tube defect, NTD)在内的中枢神经系统(central nervous system, CNS)的畸形。NTD的发生与神经干细胞的增殖、细胞的粘附分子、神经细胞的迁移等有关的分子信号的改变或作用途径被阻断密切相关。但迄今涉及上述过程的基因与具体的调控途径均知之甚少，对CDK5在脑发育早期的变化规律及其对神经干细胞的分化作用的相关报道目前尚少。因此，本研究选用正常及喂服过量维甲酸(retinoic acid, RA)诱导小鼠NTD发生模型，采用整体全胚胎原位分子杂交和免疫荧光染色、半薄切片染色、DNA芯片、脑片原位分子杂交和免疫组化染色、细胞培养、基因转染等技术；结合流式细胞检测、激光共聚焦显微镜观察、图像分析及统计学分析等手段，探讨了神经上皮中神经干细胞的分布、数量的变化及细胞周期动力学变化；比较了正常神经管形成前后基因表达的差异，分析了NTD组织与正常组织的基因表达差异谱；重点选定并观察了CDK5在脑发育中的表达变化规律，及其对神经干细胞的分化影响作用。结果如下：一、正常发育及维甲酸作用下神经上皮细胞的增殖状况1． 对孕7.5d母鼠一次性喂服50mg/kg RA后观察到E10.5d鼠胚有颅脑顶部未闭、后脑及面部异常、脊柱或尾部有裂口以及无尾或短尾等多种畸形的发生，大部分与神经系统有关。表明该剂量的RA造成了CNS发育的缺陷，该模型确实可靠。2． 采用免疫标记结合流式细胞技术、整胚Nestin免疫荧光染色结合激光共聚焦显微镜观察到正常神经上皮组织Nestin阳性细胞数目在第8.5d时最高，在E13.5d以<WP=10>后有所下降；E10.5d时 Nestin阳性细胞规律地分布于神经上皮内界膜附近。RA作用后Nestin阳性细胞数目均普遍低于正常组；尤其在E8.5d时阳性细胞数目减少最为显著 (p<0.01)；E10.5d位于内界膜处的Nestin阳性细胞明显减少且分布散乱。提示NTD的发生可能与E8.5d～9.5d时的神经干细胞减少有关。3． 采用半薄切片染色观察到正常神经管上皮中具有分裂相的细胞较多位于内界膜及套层细胞中；而RA作用后，有分裂相的细胞与同期正常相比明显减少(p<0.01)，部分区域细胞排列紊乱。流式细胞仪检测到RA作用后的神经管上皮中G0/G1期细胞百分比在E8.5d～E10.5d时明显升高(p<0.01)，S 期细胞百分比明显降低(p<0.01)。表明RA抑制了神经上皮细胞的分裂，造成G1期阻滞。RA引起NTD的发生可能与神经上皮细胞的分裂速率降低，增殖能力下降密切相关。 二、正常神经管形成及NTD发生过程中生物芯片的基因表达分析1．应用DNA微阵列技术(含1080个已知基因)，比较正常E8.5d与E11.5d神经组织基因表达，获得138个差异表达基因，上调基因71个，下调基因67个。根据基因功能分类主要包含了细胞周期与凋亡相关基因(32条)、信号转导基因(26条)、染色体与转录调控(13条)、蛋白质合成与调控(11条)、基质与骨架蛋白(14条)等几大类。其中细胞周期相关基因为最多一类，大多数呈现下调趋势，包括多个周期素家族成员(cyclinD2,cyclinA2, cyclinE1, cyclinF等)、Cdc25a、CDC5 及CDK4等，上调基因主要有p57/Kip2、CDK5等。提示包括细胞周期家族在内的多种基因参与了正常神经管关闭的生理过程。2．通过分别比较正常E10.5d与对应RA处理NTD组，正常E11.5d与对应RA处理NTD组，获得了两组NTD(也即神经胚形成缺陷)相关基因， E10.5d与E11.5d组分别有差异表达基因为20和28个。其中，E10.5d NTD组有10条和E11.5d NTD组有11条基因同时在神经胚形成前后有差异表达，为神经胚形成基因候选。NTD组还可见少数细胞周期相关基因与正常神经管形成时呈相反变化，其中CDK5在RA作用后呈现下调趋势(正常神经管形成时显示上调)。表明在神经胚形成时期CDK5的作用途径可能在RA的干扰下发生了改变。三、正常脑发育及RA作用下CDK5的表达及与其它CDKs的相关性的研究1．采用全胚胎和脑切片的原位分子杂交、免疫组化技术发现脑内CDK5mRNA从E10.5d起即可见表达，主要分布于神经上皮的套层细胞内；从E14d至成年期脑内均见CDK5mRNA与蛋白阳性信号，主要定位于神经元的胞浆与核周，于新生期前后表达达高峰；分布在包括大脑皮质、海马、丘脑、下丘脑、小脑及许多神经核团；在老年期上述区域CDK5的表达均有减弱，部分区域呈阴性。CDK5特异的激动剂p35阳性信号主要存在于神经细胞胞浆中，核不着色；p35mRNA和蛋白的表达时空模式与<WP=11>CDK5相似，于胚胎E14d已见其强阳性表达，成年期、老年期表更多还原

【Abstract】 Cyclin dependent kinase 5(CDK5) is a member of the cyclin-dependent-kinases family of serine/threonine kinases and was discovered in the early 1990s. CDK5 plays a multifunctional roles in the neurous system by virtue of phosphorylating diverse substrates. During development, it participates in neuronal proliferation, differentiation, migration, as well as axon outgrowth and synaptogenesis. Neurulation is an early developmental event that results in the formation of neural tube, the rudiment of the entire adult central nervous system(CNS). Recent studies of the developmental neurobiology have focused on the cellular and molecular mechanisms of neurulation. Findings from these studies reveal that neural stem cells are self-renewing, multipotent progenitors that give rise to the diverse types of neurons and glia. As to molecular and genomic level, the event of neural tube close involves the highly complex expression pattern and interactions of a series of genes. Neural tube defects (NTD) are one of the most common central nervous system malformation, regarded as a result caused by both environmental and genetic factors. In the recent studies, changes of the neural stem cell, extracellular matrix as well as migration signals, etc. were reported to be related with the occurrence of NTD. However, very little is known about the correlative genes and relevant pathways involving in the formation of embryonic neural tube and NTD. The roles and mechanisms of CDK5 involved in the neurogenesis, its regulative roles in the differentiation progression of neural stem cells remain largely unknown.In the present study, we established a model of retinoic acid (RA)-induced NTD through administrating RA (doses of 50mg/kg) to pregnant mice on gestational 7.5 days and harvested the normal and NTD embryo. The changes of distribution, number and cell cycle dynamics of neural stem cells during different developmental stage of the normal and the RA-treated embryo were examined using immunofluorescence on cells, whole-mount confocal of embryo by immunofluorescence labelling, semithin section staining and flow cytometry. The differentially expressed genes during the formation of embryonic neural<WP=5>tube, as well as NTD, were analyzed by mean of middle-density DNA microarrary techniques. Finally, special attentions were paid to the expression changes of CDK5 during developmental brain and the possible roles of CDK5 in differentiation of neural stem cells, by using in situ hybridization (whole-mount embryo and section), immuneohistochemistry, cell culture and gene transfection methods. The main results were as follows: Ⅰ. The cell dynamic changes of the normal and the RA-treated neuroepithelium during embryonic development 1. We established the NTD model and showed that the central nervous system malformation at age of embryonic day 10.5(E10.5) included acrania, craniofacial abnormal, spina bifida, short-tailed or tailless ect. This indicated RA-treated embryo could generate various types malformation and the model was reliable.2. Using cells immunofluorescence labeling, flow cytometry and whole-mount embryoic confocal immunofluorescence technique, higher level of the nestin positive cells were detected in both fetal cerebral vesicle and spinal cord nerve tissue than that of adult brain in normal. It reached to peak at the 8th day of embryo and decreased gradually after E13.5d. We observed that the nestin positive cells distributed mainly near the internal limiting membrane and mantle layer of neuroepithelium. The positive cells arranged regularly along the neuroepithelial layer. After RA-treated, it was found that the number of neural stem cells reduced, especially at the eighth day of embryo (p<0.01). The nestin positive cells distributed disorderly. The results suggested that the occurrence of NTD may mainly due to the number of neural stem cells reduced during formation of the neural tube.3. We observed that there were many mitotic division cells in the ventricular zone and the subplate by using semithin section stainin更多还原