NDUFA4基因缺陷导致孤立型Dandy-Walker畸形及其致病机制研究

Dandy-Walker malformation is a kind of congenital disorder which characterised by mental retardation and central nervous system damaged resulted from primary affects the cerebellum and some of its components(particularly hypoplasia of cerebellar vermis, and a cystic dilatation of fourth ventricle). There are three clinical types of Dandy-Walker malformations: Dandy-Walker syndrome，isolated Dandy-Walker malformation and Dandy-Walker variant. Unknown etiology, difficult to evaluate the prognosis and poor treatment effect are the major existing issues of isolated Dandy-Walker malformation. Our previous research on isolated Dandy-Walker malformation patients have identified and confirmed that loss-of-function variation in the NDUFA4 gene is associated with isolated Dandy-Walker malformation. Recent studies have proved that NDUFA4 gene plays significant roles in the develpoment and maintaining normal functions of cerebellum neurons, by means of regulating the mitochondrial complex IV and cytochrome C(Cytc), consequently regulating neuronal differentiation, growth and functional improvements. With the using of SD mouse neuron cells, the present study want to confirm and investigate the mechanism of NDUFA4 gene defect causing neuron cell proliferation,differentiation and apoptosis by means of overexpressing and RNAi NDUFA4 gene. And to estimate the repair effect of Cytc inhibitor in damage neuron cell causing by NDUFA4 gene defect. The implementation of this project will partialy make clear the etiology and the pathogenic mechanism for isolated Dandy-Walker malformation, and provide a theoretical basis for etiological diagnosis and treatment in this genetic CNS disease.

Dandy-Walker畸形（DWM）是一种常见的主要累及小脑和附属结构的先天性疾病。患者表现出中枢神经系统损伤等一系列临床症状。DWM分为三型，其中病因不明、预后评估困难及治疗效果差是孤立型DWM的主要特征。课题组前期研究发现和证实NDUFA4基因缺陷与孤立型DWM发生密切相关。文献资料显示，NDUFA4在神经元生长发育及维持正常功能中发挥重要作用，其机制通过调控线粒体复合物IV及细胞色素C（Cytc）在神经细胞增殖与凋亡中发挥关键作用。本课题拟利用SD大鼠神经元细胞为研究对象，通过构建NDUFA4过表达和RNAi沉默载体，探讨NDUFA4在大鼠神经元中过表达或沉默时对神经细胞增殖、分化和凋亡的影响，及对细胞色素C（Cytc）的表达调节，分析NDUFA4基因缺陷对神经元发育的调节机制，并摸索探讨Cytc抑制剂修复受损神经细胞，为阐明孤立型DWM的发病机制和潜在治疗靶点提供新的理论和依据。

Dandy-Walker 畸形（DWM）是一种主要累及小脑及其附属结构的出生缺陷疾病，病理基础是后脑发育不良和小脑中线闭合不全。本课题前期研究证实NDUFA4基因缺陷与孤立型DWM相关。因此本研究开展体外研究探讨NDUFA4基因缺陷对神经元的作用机制。.本研究通过构建NDUFA4干扰和过表达慢病毒载体，对NDUFA4进行沉默和过表达。研究结果提示，NDUFA4干扰明显抑制细胞增殖，NDUFA4过表达则促进细胞增殖。NDUFA4缺陷抑制神经元细胞增殖，细胞分化能力下降，小脑组织器官发育不良，从而导致DWM发生，尤其与该类患者中小脑蚓部发育不良相吻合。另外，NDUFA4过表达促进细胞增殖。细胞过度增殖会引起细胞分化不全。细胞过度增殖往往与肿瘤的发生密切相关，而DWM中第四脑室囊性扩大是否也是神经元过度增殖的表现，需进一步研究。.本研究提示NDUFA4基因沉默时，神经元GF、BDNF及bFGF因子表达均显著下降，提示NDUFA4与神经元的生长密切相关，NDUFA4缺陷会导致神经元生长发育障碍，证实NDUFA4在神经元生长发育中发挥着重要作用。.NDUFA4功能是构成细胞线粒体复合物IV 核心亚基，通过复合物IV 在呼吸链中发挥作用。本研究重点对Cytc以及与之相互作用的凋亡激活因子（caspase-9、caspase-3)和抗凋亡因子（Bcl-2及Bcl-xL）的变化进行动态监测。NDUFA4干扰时，Cytc（Cytc/GAPDH；0.94）和caspase-3（Caspase-3/GAPDH；0.31）显著升高，Bcl-2（Bcl-2/GAPDH;0.23)。Cytc和caspase-3 属于凋亡激活因子，这两者升高堆积时，将与各种凋亡激活因子和凋亡蛋白酶相互作用，启动凋亡级联反应，最终诱导神经元坏死，从而使小脑组织发育不良和紊乱，产生Dandy-walker表型。.本研究首次摸索应用 Cytc 抑制剂修复 神经元损伤,研究结果提示，NDUFA4干扰可抑制环孢素A对细胞凋亡的拮抗作用和降低环孢素A对神经元正向调控效应；NDUFA4过表达可促进环孢素A对细胞凋亡的拮抗作用以及提高环孢素A对神经元正向调控的效应，且这种影响作用与环孢素A浓度成正比。

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