p38有丝分裂原激酶活化参与继发性脊髓损伤后神经细胞凋亡的实验研究

THE STUDY ON PHOSPHORYLATED p38MAPK INDUCING APOPTOSIS OF NEURAL CELLS AFTER SPINAL CORD INJURY

探索脊髓损伤(SCI)后p38有丝分裂原激酶(p38MAPK)的表达及其与神经细胞凋亡的关系。采用Allen’s法建立大鼠急性SCI动物模型,用蛋白印迹法和免疫组织化学染色方法检测SCI后p38MAPK和caspase-3表达的变化;采用实时定量PCR法检测SCI后caspase-3 mRNA的表达;用原位末端标记法(TUNEL)检测SCI后神经细胞凋亡。结果表明SCI后总p38MAPK表达无变化,但p38MAPK磷酸化明显增多,于伤后6h达高峰;伤后24hcaspase-3表达明显增加。TUNEL检测显示,伤后6h受损伤的脊髓组织有少许凋亡细胞出现,24h细胞凋亡最明显。鞘内注射p38MAPK抑制剂SB203580,明显减少caspase-3的表达,并减少细胞凋亡。以上结果显示SCI后损伤局部p38MAPK激活诱导了caspase-3基因表达,导致神经细胞凋亡;抑制p38MAPK可以阻止SCI后继发的神经细胞凋亡。

To investigate the changes of p38 mitogen activated protein kinase （MAPK） after spinal cord injury （SCI）, and whether the changes are involved in apoptosis of neural cells. The model of SCI was made with Allen＇s method. The expression of p38MAPK and easpase-3 after SCI was tested with Western blot and staining, and caspase-3 mRNA was tested using real time reverse transcription polymerase chain reactions （ real time RT-PCR）. Neuron apoptosis was assessed with the terminal deoxynucleotidyl deoxyuredine triphosphate-biotin nick end labeling （TUNEL） method. The expression of p38MAPK had not change while that of the phosphorylated p38MAPK was increased significantly after injury, with a peak in expression levels within 6 h after injury. By 24 h after injury, caspase-3 expression was increased markedly in the injured spinal cord. TUNEL-positive cells were first observed in the lesion area 6 h after SCI. The largest number of TUNEL-positive cells was observed at 24 h after SCI. Intrathecal injection of the p38 MAPK inhibitor SB203580 can reduce the expression of caspase-3 significantly and the number of TUNEL-positive cells in the injured spinal cord. Our present study indicates that the increased phosphorylation of p38MAPK plays an important role in induction of neuron apoptosis through caspase-3 after SCI,and SB203580 can reduce the apoptosis of neuron after injury.