ANALYSIS OF MECHANISMS OF NEURONAL CELL DEATH USING ADENOVIRUS VECTOR SYSTEM

本课题的目的是建立能够将基因导入鸡胚神经细胞的腺病毒载体系统，并在体内外研究Bcl-2和Crm A对神经细胞死亡的影响。主要结果如下：（1）在第一次实验中，我们未能获得CAG启动子调控下表达Bcl-2的重组腺病毒。利用Cre-LoxP系统对Bcl-2基因表达进行调控，成功获得了表达Bcl-2的重组腺病毒。(2)证实腺病毒载体介导的基因在体外培养的鸡背根节神经元和运动神经元中表达。在体外导入人Bcl-2可使DRG神经元和运动神经元免于细胞死亡。(3)同时也证实了腺病毒载体注射入腿芽后可逆行转运至外侧运动柱的运动神经元。Bcl-2通过这种逆行转运的方式被引入，可以在肢芽切除后挽救一些运动神经元。(4)在大鼠C6胶质瘤细胞系系统中，腺病毒载体介导的人Bcl-2诱导细胞死亡。(5)腺病毒载体介导的Crm A基因对DRG神经元系统无明显的拯救作用。

Aims of this project were (I) to establish adenovirus vector system that is capable of introducing genes into neuronal cells of the chick embryo, and (II) to analyze effects of Bcl-2 and Crm A on neuronal cell death in vitro and in vivo. Major results are the following :(1) In the first trial, we failed to obtain recombinant adenovirus that express Bcl-2 under regulation of CAG promoter. Employing Cre-LoxP system that can regulate expression of genes, we succeeded to obtain the recombinant adenovirus that express Bcl-2.(2) It was confirmed that genes introduced by adenovirus vecotrs were expressed in the chick DRG neurons and chick motoneurons in vitro. Introduced human Bcl-2 could rescue DRG neurons and motoneurons from cell death in vitro.(3) It is also confirmed that adenovirus vectors injected into leg bud were retrogradely transported to the motoneurons in the lateral motor column. Bcl-2 introduced with this retrograde transport could rescue some motoneurons after limb bud removal.(4) In the rat C6 glioma cell line system, human Bcl-2 introduced by adenovirus vector induced cell death.(5) We could not find rescuing effects of Crm A gene introduced by adenovirus vector in the DRG neuron system.