肌萎缩侧索硬化脊髓器官型培养模型的建立  

Establishment of Organotypic Culture Model of Spinal Cord for Amyotrophic Lateral Sclerosis

作　　者：刘卫刚[1] 王晓娟[2] 肖向建[3] 马征[1] 宋学琴[1] 王丽琴[1] 李春岩[1] 

机构地区：[1]河北医科大学第二医院神经内科,石家庄050000 [2]北京同仁医院神经科,北京100730 [3]河北省人民医院神经科,石家庄050000

出　　处：《细胞生物学杂志》2004年第6期635-639,共5页Chinese Journal of Cell Biology

基　　金：河北省自然科学基金资助项目(No.303487)

摘　　要：利用谷氨酸转运体抑制剂苏-羟天冬氨酸(THA)制备选择性运动神经元凋亡的肌萎缩侧索硬化(ALS)脊髓器官型培养模型。取出生后8天乳鼠腰段脊髓组织切成脊髓薄片,在培养液中分别加入不同浓度THA,用SMI-32免疫组化染色对脊髓腹角α运动神经元进行鉴定,calretinin免疫组化染色对背角中间神经元进行鉴定,测定培养液中谷氨酸(Glu)、乳酸脱氢酶(LDH)的含量,并与对照组比较。结果显示对照组α运动神经元数目恒定;THA引起培养液中剂量依赖性Glu、LDH 含量增高和SMI-32阳性的α运动神经元数目减少,脊髓背角的中间神经元损伤相对较轻;100 μmol/L THA组在体外培养4周后,细胞外Glu含量增高,SMI-32阳性的α运动神经元数目较对照组明显减少,背角的中间神经元数目无显著变化,可以制成ALS脊髓器官型培养模型。In this study, we developed a cultured organotypic spinal cord model of selective motor neuron death for amyotrophic lateral sclerosis (ALS), using threohydroxyaspartate (THA), a inhibitor of glutamate transport. Cultured organotypic spinal cord slices were prepared from lumbar spinal cords of 8-day-old rat pups. Various concentrations of THA were continuously added into the culture medium. Ventral α-motor neuron survival was evaluated by culture morphology and SMI-32 immunohistochemistry staining. Interneurons in dorsal horn were identified by calretinin immunohistochemistry staining. Glutamate (Glu) and lactate dehydrogenase (LDH) levels in culture medium were measured. The results showed that the spinal cord slices in the control group could maintain excellent organotypic cellular organization and a stable population of ventral SMI-32 positive α-motor neurons. THA could produce a slow dose-dependent loss of SMI-32 positive α-motor neurons and a elevation of Glu , LDH levels in culture medium. After the slices cultured 4 weeks in vitro, 100 μmol/L THA resulted in the loss of SMI-32 positive α-motor neurons, increase of Glu level and no significant change of inter-neurons in the dorsal horn, which could be acted as an effective organotypic culture model of ALS.

关 键 词：肌萎缩侧索硬化 脊髓 器官型培养模型 苏-羟天冬氨酸 谷氨酸 乳酸脱氢酶 神经系统变性疾病 运动神经元 

分 类 号：R-332[医药卫生]