水通道蛋白4在小鼠脑发育过程中的表达及其在维持成年小鼠室管膜完整性中的作用

Expression of Aquaporins-4 and Its Relation to Brain Water Content in the Developmental Mouse Brain

作者: 专业:药理学 导师:肖明 年度:2009 学位:硕士  院校: 南京医科大学

Keywords

aquaporin-4, brain water content, development, knockout, water homeostasis, connexin43, ependymal cells, gap junction, gene knockout

        研究显示水通道蛋白4(AQP4)参与脑水平衡的形成和维持。然而,在发育过程中整个脑区AQP4的表达变化,及其与脑含水量改变的相关性还未见报道。Western blot结果和光密度分析显示CD1小鼠在生后第1周脑内AQP4的表达水平仅占成年水平的21.3%,但是在第2周AQP4表达显著性升高,达到成年水平的67.4%,到第4周达到成年水平的88.6%。与AQP4表达增加相一致,在发育过程中脑含水量动态降低。统计学分析证实发育过程中AQP4蛋白表达水平和脑含水量呈部分负相关关系(rP2P = 0.92, P = 0.002)。AQP4基因敲除鼠和野生型小鼠在生后第1周内脑含水量没有显著性差别。然而,从第2周至成年期,AQP4基因敲除鼠基础脑含水量较同窝对照组增高。此外,我们应用免疫组织化学结合半定量图像分析研究了CD1小鼠出生后脑中AQP4的时空表达变化。结果显示在脑干和下丘脑AQP4表达比在大脑皮层和小脑要早。至出生后第14天,上述脑区血管周围星形胶质细胞突起和室管膜细胞基底外侧膜上AQP4的表达均呈现成熟特征。这些结果揭示了脑中AQP4的发育特征,为AQP4在脑水平衡稳态的成熟建立中发挥重要作用提供直接证据。室管膜细胞为沿脑室壁排列的一类上皮细胞,参与构成脑-脑脊液屏障和脑脊液的形成。研究表明在室管膜细胞的基底外侧膜上表达对水具有快速转运作用的水通道蛋白4(AQP4)。AQP4特异性表达特征提示其可能参与维持室管膜结构和功能的完整性。本学位论文应用本实验室自行构建的成年AQP4基因敲除鼠作为研究对象,来验证此推测。组织学分析显示AQP4敲除鼠侧脑室和导水管的室管膜层结构紊乱。92.7%的AQP4敲除鼠表现出侧脑室容积的降低,而7.3%显示扩大或正常的侧脑室,伴有狭窄的导水管。免疫组织化学结果显示AQP4缺失导致室管膜细胞间缝隙连接蛋白43(Cx43)表达的降低。电镜结果进一步证实AQP4敲除鼠室管膜细胞基底外侧膜上连接复合体缺失。此外,与野生型小鼠相比,AQP4敲除鼠脑脊液生成减少而脑含水量增加。上述结果提示AQP4在维持室管膜结构和功能方面发挥重要的作用。另外,AQP4敲除导致的脑室表型异常的个体差异性提示AQP4功能具有多态性。
    Previous studies indicate that water transport protein aquaporin-4 is involved in the formation and maintenance of brain water homeostasis. Nevertheless, developmental feature of aquaporin-4 in the whole brain and its relation to brain water content during postnatal life remains unknown. Western blot and densitometry analysis showed that aquaporin-4 expression in CD1 mouse brain was only 21.3% of the adult level one week after birth, but significantly increased to 67.4% at the second week, and reached 88.6% of adult level by 4 weeks. Consistent with increased aquaporin-4 expression, dynamic reduction in brain water content occurred during postnatal life. Statistical analysis demonstrated a negative partial correlation between aquaporin-4 expression and brain water content levels during postnatal development (rP2P = 0.92 and P = 0.002). There was no difference in brain water content between aquaporin-4 null and wild-type mice at the first week after birth. However, aquaporin-4 null mice showed greater brain water content compared with littermate controls from the second week up to adult stage. Moreover, we investigated the spatio-temporal immunolocalization of AQP4 in postnatal CD1 mouse brain. Semiquantitative image analysis revealed that AQP4 expression in the brain stem and hypothalamus was earlier than that in the cerebral cortex and cerebellum. Mature feature of AQP4 localization in perivascular astrocyte processes and basal lateral membrane of ependymal cells was completed at approximately 2 week after birth. These results characterize developmental feature of aquaporin-4 in the postnatal brain and highlight a vital role of aquaporin-4 in the maturation of brain water homeostasis. Ependymal cells form the walls of the ventricles, and take part in the production of cerebrospinal fluid. Aquaporin-4 (AQP4), one isoform of water channels mainly distributed in the brain, is restricted to basolateral plasma membranes of ependymal cells. The highly polarized expression of AQP4 suggests it may be involved in maintaining the structural and functional integrity of the ependyma. This hypothesis was validated by use of adult AQP4 knockout mice generated by Fan et al. (J Neurosci Res. 2005 Nov 15;82(4):458-64). Histological analysis showed disorganized ependymal layer of the lateral ventricle and aqueduct in AQP4-deficiency mice. A majority (92.7%) of null mice displayed reduced lateral ventricular volume, while a small fraction (7.3%) had enlarged or normal ventricular size with a narrow aqueduct. Immunohistochemistry demonstrated that AQP4 deletion resulted in decreased expression of gap junction protein connexin43 in the ependymal cells. Electron microscopy confirmed junctional complex absence at basolateral membranes of ependymocytes. Moreover, AQP4 knockout mice showed decreased cerebrospinal fluid production and increased brain water content compared with wild-type mice. These results highlight a key role of AQP4 in maintaining the structure and function of the ependyma. In addition, variable profiles of ventricle system in adult AQP4 null mice indicate functional AQP4 polymorphisms.
        

水通道蛋白4在小鼠脑发育过程中的表达及其在维持成年小鼠室管膜完整性中的作用

英文缩略词表4-5
第一部分 水通道蛋白4 在小鼠脑发育过程中的表达及其与脑含水量的相关性5-25
    中文摘要5-6
    英文摘要6-8
    前言8
    材料与方法8-10
    结果10-18
    讨论18-21
    参考文献21-25
第二部分 水通道蛋白4 在维持成年小鼠室管膜完整性中的作用25-51
    中文摘要25-26
    英文摘要26-27
    前言27-28
    材料与方法28-31
    结果31-39
    讨论39-44
    参考文献44-51
小结51-52
综述52-66
    参考文献60-66
附录66-67
    攻读学位期间发表文章情况66-67
    致谢67
        下载全文需10


本文地址:

上一篇:Aquaporin4基因敲除对吗啡诱导条件性位置偏爱及抑制神经再生的影响
下一篇:MicroRNAs及其靶基因在葡萄膜黑色素瘤和胃癌发生发展中的意义

分享到: 分享水通道蛋白4在小鼠脑发育过程中的表达及其在维持成年小鼠室管膜完整性中的作用到腾讯微博           收藏
评论排行
公告