Nestin在高眼压大鼠视网膜神经胶质细胞中的表达

摘要

Background Elevated intraocular pressure leads to the loss of retinal ganglion cells and vigorous reaction of retinal glial cells.The expression of nestin in retinal glial cells secondary to hypertention and its significance are unclear.ObjectiveThis study aim to investigate the expression of nestin in retinal glial cells (RGCs) in ocular hypertention rats.Methods The ocular hypertention models were established by cauterizing the limbus-draining veins in the right eyes of 42 SD rats,and a conjunctival incision in the left eyes of the rats served as the sham group.The intraocular pressure (IOP) was measured with the Tono-Pen XL tonometer.The number of RGCs in the rats with ocular hypertention was counted.The expression of the nestin protein in RGCs was semi-quantitatively analyzed using Western by immunochemistry.Double immunofluorescence was carried out to evaluate the the confocal laser scaning microscope.Results Significant differences were found in the IOP between the model group and the sham group at various time points (P＜0.05).In 1 week to 3 weeks after operation,the number of RGCs significantly declined in the model group compared with the sham group (P＜0.05).Immunochemistry showed that from 2 hours through 1 week after operation,the expression of nestin was gradually enhanced in the model group in comparison with the sham group.Western blot revealed that the expression of the nestin protein reflected a similar tendency to that of immunofluorescence.The increased introcular pressure as manifested by the induced expression of nestin.Immunoelectron microscopy also confirmed the induced expression of nestin especially at their end-feet suggests a potential neuroprotective mechanism in neuronal degeneration.Nestin may be a useful biomarker for retinal injury study.%目的 探讨第六类中间丝蛋白nestin在高眼压大鼠视网膜神经胶质细胞中的表达情况及意义.方法 42只SD大鼠采用烧灼右眼涡静脉的方法 制作高眼压模型为模型组,剪开左眼结膜作为假手术组.测量并记录眼压,计数术后不同时间点视网膜神经节细胞(RGCs)数.Western blot半定量分析术后视网膜内nestin蛋白含量.免疫电镜检测nestin在高眼压大鼠视网膜Müller细胞上的诱导表达.行nestin/谷氨酰胺合成酶(GS)或nestin/胶质纤维酸性蛋白(GFAP)免疫荧光双标,共焦显微镜观察nestin/GFAP在Müller细胞上的表达情况.结果 术后各时间点模型组与假手术组眼压的差异有统计学意义(P＜0.05).术后1～3周模型组RGCs数量显著减少,与假手术组比较差异有统计学意义(P＜0.05).免疫荧光结果 显示术后2h～3周,模型组nestin在Müller细胞上表达的荧光强度逐渐增强,与假手术组比较差异有统计学意义.Western blot半定量分析结果 与免疫荧光染色结果 一致.免疫电镜结果 进一步证实眼压升高后Müller细胞上出现nestin的诱导表达.结论 Nestin在Müller细胞上的诱导表达是Müller细胞对眼压升高产生的一种反应,nestin的表达量与病程进展相一致.眼压升高时,nestin在Müller细胞上诱导表达,尤其在足板处的强烈表达,可能对RGCs具有保护作用.Nestin有望成为一种新的研究视网膜损伤的生物标记物.