【作者】 张涛；

【导师】 高国栋；

【作者基本信息】 第四军医大学 ， 外科学， 2009， 博士

【摘要】 CD226分子是一种分子量为~65kDa的跨膜糖蛋白,属于免疫球蛋白超家族成员,是一种细胞粘附分子(cell adhesion molecule,CAM)。自1985发现该分子以来,人们对CD226分子的基因、结构、表达、分布及功能做了大量研究,并且证实该分子广泛参与机体免疫系统多种重要的生物学功能,包括T细胞的增殖与分化,血小板的活化与凝集,NK细胞的杀伤等。然而,有关机体免疫系统以外CD226分子表达和功能的研究却未见报道。近些年来,越来越多的研究表明许多本来表达于免疫系统的CAM也表达和分布于神经系统,并且与中枢神经系统(central nervous system,CNS)内突触发生和突触结构可塑性调节等功能密切相关。CAM是一种广泛分布于机体内的跨膜糖蛋白,在细胞间的识别与粘附过程中发挥重要作用,进而广泛参与机体多种重要的生理功能,包括胚胎发育、细胞信号转导、炎症及肿瘤转移等等。近些年来,包括neurexin、neuroligin、cadherin、Nectin、NCAM(neural cell adhesion molecule,神经细胞粘附分子)以及SynCAM在内的一些CAM家族成员,已经被证实表达和分布于机体神经系统内,并且在突触发生过程中的靶向识别、突触分化、以及学习记忆过程中突触的可塑性调节方面发挥了重要作用。迄今为止,有关CD226分子在神经系统表达和分布的研究尚未见报道,但是越来越多的证据显示CD226分子可能分布于CNS内并且参与重要的生理功能。首先,CD226分子与其他分布于神经系统内的CAM分子结构类似,表现为这些分子的胞膜外区都有若干个免疫球蛋白超家族V样结构域,胞浆区末端均有一段能够与细胞骨架特定结构结合的保守序列。其次,最近的研究表明Nectin家族成员Nectin-2/CD112及Nectin样分子(Nectin-like molecule,Necl)家族成员Necl-5/CD155是CD226的配体,而Nectin及Necl家族中的不少分子已被证实表达于神经系统,并且分别分布于特定脑区的突触前膜和/或突触后膜。本研究综合运用分子生物学、免疫学及形态学等多种研究方法,在国际上首次证实了CD226分子在小鼠神经系统内的表达和分布以及该分子在小鼠生后发育过程中表达和分布的变化;证实了在体外培养条件下,外源性CD226及CD155分子能够显著促进海马神经元的生长发育,并且在神经突起生长过程中发挥了靶向识别作用;证实了缺氧不同时间后海马神经元内CD226分子表达水平逐渐下调,且缺血性脑血管病患者及脑肿瘤患者血清中可溶型CD226分子水平发生上调。具体研究内容如下:1.首先运用RT-PCR及Western blotting方法,从mRNA和蛋白水平证实了CD226分子主要表达于小鼠脑内的海马和小脑组织;免疫组织化学结果显示,在成年小鼠脑内CD226分子主要分布于海马CA3区透明带及邻近的齿状回门区,小脑皮质颗粒细胞间隙以及Purkinje细胞胞体,荧光双标结果显示CD226分子与突触标志物Synaptophysin、Syntaxin以及PSD-95广泛共存,而与神经元标志物NeuN以及星形胶质细胞标志物GFAP(glial fibrillary acidic protein,神经胶质酸性蛋白)没有共存,免疫电镜结果进一步证实CD226分子可能分布于上述脑区的突触前膜和突触后膜;而在小鼠生后发育早期,P12以前CD226分子并不表达于其成年时分布脑区,而是一过性表达于与上述脑区具有密切纤维联系的邻近或者远离的神经核团内。2.成功对新鲜分离的胎鼠海马神经元细胞进行体外培养,并且运用免疫荧光染色方法观察了体外培养不同时间海马神经元的细胞形态以及CD226分子的表达情况;运用脂质体转染的方法,成功地将mCD22(6murine CD226,小鼠CD226)-GFP或mCD155(murine CD155,小鼠CD155)-RFP两种质粒转染入Hela细胞,并将其与新鲜分离的胎鼠海马神经元细胞进行共培养,进而证实在体外培养条件下,外源性CD226及CD155分子能够显著促进海马神经元的生长发育,主要表现为神经突起数量增多和长度增加,而且外源性CD226及CD155分子还能促进神经突起向表达它们的Hela细胞进行生长并且发生纤维联系,发挥了靶向识别作用。3.成功建立新鲜分离的胎鼠海马神经元细胞缺氧模型,运用免疫荧光染色方法观察了细胞缺氧不同时间后神经元形态的改变及CD226分子表达的变化;运用Western blotting方法证实了随着细胞缺氧时间的延长,神经元内CD226分子的表达水平逐渐下调,缺氧24h后几乎完全消失。运用改良的夹心酶联免疫吸附实验(enzyme linked immunosorbent assay,ELISA)证实,缺血性脑血管病患者及脑肿瘤患者血清中可溶型CD226分子的水平显著高于正常人。总而言之,本次实验在国际上首次证实了CD226分子表达和分布于神经系统,并且对CD226分子在神经元生长发育过程中发挥的作用、以及疾病时CD226分子表达水平的变化进行了初步探讨,为进一步研究CD226分子在神经系统内可能参与的生理功能及其分子机制奠定了基础。更多还原

【Abstract】 CD226, a member of immunoglobulin superfamily, is a ~65kDa transmembrane glycoprotein. As a cell adhesion molecule (CAM), CD226 has been widely studied about its gene, structure, expression and localization in the immune system and has been identified to be involved in several important biological functions including T cell proliferation and differentiation, platelet activation and aggregation, NK cell cytotoxicity and so on. However, the presence of CD226 in the other systems beside immune system has not been reported yet. But recently, more and more members of CAM, which were initially present in the immune system, have been identified to be present in the central nervous system (CNS) and play important roles in synaptogenesis and synaptic plasticity.CAMs are mainly membrane glycoproteins that mediates cell-cell adhesion and play essential roles in various functions, including embryonic development, cell signal transduction, inflammation, and tumor metastasis. During the last two decades, an increasing number of the CAM family members, such as neurexin, neuroligin, cadherin, Nectin, NCAM(neural cell adhesion molecule), and SynCAM, have been demonstrated to be located in the CNS and play important roles not only in initial target recognition as well as in synapse specification and maturation during synaptogenesis, but also in synaptic plasticity during learning and memory processes.Although the expression of CD226 in the CNS has not been reported yet, several lines of evidence have indicated that CD226 might be present and have important roles in the CNS. As a member of the immunoglobulin superfamily, CD226 possesses the typical structure of the other identified members of CAM reported in the CNS: single-spanning membrane proteins with several Ig(immunoglobulin)-like extracellular domains and an intracellular tail containing a PDZ-binding motif characterized by a conserved sequence. Furthermore, CD226 has recently been identified as the receptor for Nectin-2/CD112 and Necl (Nectin-like molecule) family member Necl-5/CD155 while many Nectin and Necl family members have been identified to be localized on the pre- and post-synaptic membrane.Molecular biological, immunological and morphological methods have been employed in our present research and the major achievements are: CD226 has been identified to be expressed and localized in the murine brain during adulthood and postnatal development. Exogenous CD226 and CD155 would not only evidently promote the growth and development of hippocampal neurons but also assist target recognition during neurite growth in vitro. The level of CD226 in the hippocampal neurons gradually decreased after hypoxia while the level of soluble CD226 in the serum of cerebral ischemic and tumor patients increased. The details are as following:1. CD226 mRNA and protein in the murine hippocampus and cerebellum was detected by RT-PCR and Western blotting, respectively. Furthermore, immunohistochemical staining revealed that CD226 was mainly distributed in the hilus of the dentate gyrus and stratum lucidum aligned along the pyramidal cells in the hippocampal CA3 area, the interspaces of granular cells and the somata of the Purkinje cells in the cerebellar cortex during adulthood. Double-staining results revealed that CD226 co-localized well with synaptic marker proteins including Synaptophysin, Syntaxin and PSD-95 while it didn’t co-localize with NeuN or GFAP (glial fibrillary acidic protein). Ultrastructural analysis revealed that CD226 was located both on the pre-synaptic membrane and post-synaptic membrane. During postnatal development, CD226 could not be detected at its adult locations until postnatal day 12; however, it was temporally expressed in the somata of neighboring or distant nuclei associated with its adult location.2. Freshly dissected hippocampal neurons of embryonic mouse were successfully cultured in vitro. Immunofluonrescent staining was employed to observe the CD226 expression in the hippocampal neurons at different time point during culture in vitro. Two different plasmids, mCD226(murine CD226)-GFP or mCD155(murine CD155)-RFP, was successfully transfected into Hela cells by liposome transfection method. Then the transfected Hela cells were co-cultured with the freshly dissected hippocampal neurons of embryonic mouse. The co-culture of transfected Hela cells and neurons indicated that exogenous CD226 and CD155 would not only evidently promote the growth and development of hippocampal neurons by increasing the quantity and length of the neurite but also assist target recognition for the neurite by guiding them to head for the transfected Hela cells and establish fiber connections. 3. Hypoxia model of freshly dissected hippocampal neurons was successfully established and immunofluonrescent staining was used to observe the morphological changes of the hippocampal neurons and CD226 expression in them at different time point after hypoxia. Western blotting analysis revealed that CD226 expression in the hippocampal neurons was gradually down-regulated after hypoxia and vanished at 24h after hypoxia. Sandwich enzyme linked immunosorbent assay (ELISA) results indicated that the soluble CD226 in the serum of cerebral ischemic and tumor patients was evidently up-regulated when compared with that of the healthy control.In conclusion, our present study identified the expression and distribution of CD226 in the CNS for the first time, investigated the possible role of CD226 during the growth and development of hippocampal neurons and demonstrated the changes of CD226 expression in different diseases, which established the foundation for further exploration of the physiological functions and molecule mechanism of CD226 in the CNS.更多还原