【摘要】 目的研究体外培养大鼠星形胶质细胞划痕损伤后水通道蛋白4(aquaporin4,AQP4)表达变化,并观察丝裂原活化蛋白激酶(mitogen-activated protein kinases,MAPKs)信号通路特异性抑制剂对此变化的影响。方法利用传代培养10d左右的大鼠星形胶质细胞制作划痕损伤模型,部分实验于划痕损伤前1h分别加入ERK抑制剂U0126,p38MAPK抑制剂SB203580及JNK抑制剂SP600125。观察细胞形态变化、GFAP免疫细胞化学染色变化,并用实时荧光定量PCR法检测AQP4mRNA表达变化。结果划痕损伤后12h,细胞形态由扁平形变为不规则形,部分细胞突起向划痕区伸出,划痕区出现GFAP免疫化学染色阳性细胞,AQP4mRNA表达水平明显降低(P<0.05),而U0126,SB203580和SP600125预处理均能阻止此种变化(P<0.05)。结论划痕损伤引起培养星形胶质细胞AQP4mRNA表达下调,MAPKs抑制剂可对抗此作用,提示MAPKs信号通路参与星形胶质细胞损伤后AQP4表达变化的调节。更多还原

【Abstract】 Objective Aquaporin 4(AQP4) is the predominant water channel protein in the brain,and plays important roles in the development of brain edema.However the mechanisms regulating changes of AQP4 expression after brain injury are not known.In this study,we investigated the change of AQP4 expression in cultured rat astrocyte after scratch injury and explored the effects of pre-treatment with mitogen-activated protein kinases(MAPKs) inhibitors.Methods Secondary cultured astrocyte at 10 days were used to prepare scratch injury model,the morphologic change of astrocyte was observed through phase contrast microscope,and immunostaining of anti-glial fibriliary acidic protein(GFAP) was performed.Meanwhile,the AQP4 mRNA level in astrocyte was determined by real-time fluorescent quantitative reverse transcription polymerase chain reaction.Results Without injury the cultured astrocyte were uniformly thin and flat,while 12 h after scratch injury,the astrocyte became irregularly shaped and the cell processes along the margin of the wound extended to the cell-free area.Also,the immunostaining showed stronger GFAP staining at 12 h after injury,and hypertrophic astrocyte occurred.The level of AQP4 mRNA in astrocyte decreased significantly at 12 h after scratch injury(P<0.05),which was antagonized by pretreatment with ERK inhibitor U0126,p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125 respectively(P<0.05).Conclusion Our results demonstrate that scratch injury on astrocyte directly down-regulated AQP4 mRNA expression,and MAPKs inhibitors can antagonize the response.These findings suggest that MAPKs signal transduction pathway may be involved in the regulation of AQP4 expression after injury.更多还原