大鼠选择性运动神经元死亡的脊髓器官型培养模型的建立  

Establishment of rat organotypic culture model for selective motor neuron death

作　　者：肖向建[1] 王晓娟[2] 刘卫刚[1] 李春岩[1] 

机构地区：[1]河北医科大学第二医院神经内科 [2]北京同仁医院神经科,北京100730

出　　处：《基础医学与临床》2004年第6期687-691,共5页Basic and Clinical Medicine

基　　金：河北省自然科学基金 (30 3487)

摘　　要：利用谷氨酸转运体抑制剂苏 羟天冬氨酸 (threo hydroxyaspartate ,THA)制备大鼠选择性运动神经元死亡的脊髓器官型培养模型。取出生后 8天乳鼠的腰段脊髓组织切片做脊髓器官型培养 ,在培养液中分别加入不同浓度THA(5 0 μmol/L、10 0 μmol/L、5 0 0 μmol/L) ,用神经元的特异性免疫组化染色SMI 32对脊髓腹角α运动神经元及背角中间神经元计数 ,测定培养液中乳酸脱氢酶 (LDH)的含量 ,并与对照组做比较。结果显示对照组α运动神经元数目恒定 ,THA可以引起剂量依赖性的α运动神经元数目减少和培养液中LDH含量增高 ,其中加用THA 10 0 μmol/L后 3周后即有α运动神经元数目较对照组明显减少 ,而脊髓背角的中间神经元数目无显著差异。利用谷氨酸转运体抑制剂THA 10 0 μmol/L可以制成选择性运动神经元损伤模型 ,为进一步研究肌萎缩侧索硬化 (ALS)的发病机制及探讨神经保护治疗提供了有效的方法。To develope a model for selective motor neuron death in cultured organotypic spinal cord using threohydroxyaspartate(THA), an inhibitor of glutamate transport carrier. Organotypic spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentrations of THA were added into the culture medium respectively.Ventral α-motor neurons’ and dorsal interneurons’ survivals were evaluated by culture morphology and by monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, immunohistochemical staining and compared with the controls. Lactate dehydrogenase (LDH) level in culture medium was also measured. The results showed that the spinal cord explants in control group could maintain excellent organotypic cellular organization and a stable population of ventral α-motor neurons. THA could produce a slow and a dose-dependent loss ofα-motor neuron and a increase of LDH enzyme activity in culture medium. 100μmol/L THA resulted in significant decrease of α-motor neuron after 3 weeks’ culture. In contrast the inter-neurons in the dorsal horn were less affected. Treatment with THA (100μmol/L) could cause selective α-motor neuron death, which provides an effective organotypic culture model for studying pathogenesis and neuroprotection of amyotrophic lateral sclerosis.

关 键 词：运动神经元 器官型培养 脊髓 对照组 大鼠 中间神经元 死亡 数目 乳酸脱氢酶(LDH) 培养液 

分 类 号：R-332[医药卫生]