谷氨酸转运抑制剂对器官型培养脊髓片的影响  

EFFECTS OF GLUTAMATE TRANSPORT INHIBITOR ON ORGANOTYPIC CULTURED SPINAL CORD SLICES

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

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

出　　处：《实验生物学报》2005年第2期171-176,共6页Acta Biologiae Experimentalis Sinica

基　　金：河北省自然科学基金(303487)

摘　　要：观察谷氨酸转运体抑制剂苏-羟天冬氨酸(Threo-hydroxyaspartate,THA)对器官型培养的脊髓片的影响,探讨谷氨酸在运动神经元损伤中的作用。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,在培养液中加入不同浓度THA(50μmol/L、100μmol/L、500μmol/L),用神经元的特异性免疫组化染色剂SMI-32,非磷酸化神经丝标记物,对脊髓腹角α运动神经元进行鉴定,用单克隆抗钙网膜蛋白(calretinin)抗体对背角中间神经元进行记数,测定培养液中乳酸脱氨酶(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可以引起剂量依赖性的培养液中LDH含量增高和α运动神经元数目减少,而脊髓背角的中间神经元损伤相对较轻,其中THA100μmol/L组在体外培养4周后出现类似于肌萎缩侧索硬化(ALS)的病理改变: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变化。细胞外谷氨酸增高主要对运动神经元造成损伤,脊髓运动神经元较感觉神经元对谷氨酸的兴奋毒作用更加敏感。This study was aimed at investigating the effect of glutamate on motor neurons in organotypic cultured spinal cord slices treated by threohydroxyaspartate (THA), an inhibitor of glu- tamate transporter. The spinal cord cultures were prepared using lumbar spinal cord slices from 8-day-old rat. Various concentration of THA(50μmol/L, 100μmol/L,500μmol/L)was added into the culture medium respectively. Ventral α-motor neurons survival was evaluated by immunohisto- chemistry staining monoclonal antibody SMI-32, a nonphosphorylated neurofilament marker, and in- terneurons in dorsal horn were identified by monoclonal anti-calretinin antibody staining. Lactate dehydrogenase (LDH) level in the culture medium was also measured. The spinal cord slices in the control group could maintain excellent organotypic cellular organization and a stable population of ventral α-motor neurons. THA caused a slow dose-dependent loss of α-motor neurons and an increase in LDH enzyme activity in the culture medium while dorsal interneurons were less dam- aged. 100μmol/L THA resulted in a significant decrease in α-motor neurons after cultured for 4 weeks. On the contrary, the interneurons in the dorsal horn were less affected. It was also ob- served in patients with amyotrophic lateral sclerosis (ALS). Excellular Glu mainly caused selective a-motor neuron death, and motoneurons were more sensitive to glutamate excitotoxicity than sen- sory neurons in the spinal cord.

关 键 词：抑制剂 器官 脊髓运动神经元 神经元损伤 谷氨酸转运体 脊髓背角 乳酸脱氢酶 感觉神经元 MOL 培养液 天冬氨酸 组织切片 免疫组化 病理改变 体外培养 元数 染色剂 特异性 标记物 神经丝 磷酸化 中间 膜蛋白 单克隆 对照 

分 类 号：R96[医药卫生—药理学]