分泌型卷曲相关蛋白4在肌萎缩脊髓侧索硬化症转基因鼠脊髓中的表达变化

目的 探讨分泌型卷曲相关蛋白4(sFRP4)在成年肌萎缩脊髓侧索硬化症(ALS)转基因模型鼠脊髓内的表达变化,及sFRP4在ALS发病中的作用.方法 选取ALS转基因鼠和同窝野生型鼠各30只,分别于鼠95d、108d和122d取材,部分鼠经4%多聚甲醛心脏灌注固定,分离脊髓和制备冷冻切片,部分鼠分离脊髓,提取RNA和蛋白.分别应用免疫组织化学染色技术、RT-PCR和Western blotting检测sFRP4 mRNA和蛋白水平在不同时间点的变化情况.结果 与同窝野生型鼠比较,ALS转基因鼠sFRP4 mRNA和蛋白表达在95d、108d和122d均升高(P< 0.05).免疫组织化学结果 显示,sFRP4阳性细胞主要分布在脊髓灰质前角,白质轴突呈阳性反应,与同窝野生型鼠比较,ALS转基因鼠sFRP4阳性细胞明显增多.免疫荧光双标结果 显示,sFRP4标记的阳性细胞共表达胶质纤维酸性蛋白和β-tubulin Ⅲ,ALS转基因鼠sFRP4/GFAP双阳性细胞增多.结论 在ALS转基因鼠发病脊髓中sFRP4阳性细胞明显增多,sFRP4 mRNA和蛋白表达升高,表明sFRP4与肌萎缩脊髓侧索硬化症的发生密切相关.

Expression change of the secreted frizzled-related protein 4 in spinal cord of amyotrophic lateral sclerosis transgenic mice

Objective To study the expression change of sFRP4 in the spinal cord of adult amyotrophic lateral sclerosis(ALS) transgenic mice, and explore the role of sFRP4 in the pathogenesis of ALS. Methods Both the ALS transgenic mice and wild-type mice were selected, some were perfused intracardially with 4% paraformaldehyde at the age of 95days, 108days and 122days.Their spinal cords were dissected and sectioned. Some mice were anesthetized and spinal cords were quickly removed. RNA and total protein were obtained. The distribution of sFRP4 positive cells, and expression changes of sFRP4 mRNA and protein in the spinal cord of adult ALS transgenic mice and wild-type mice were observed by immunohistochemical techniques, RT-PCR and Western blotting at different time points. Results Compared with wild-type mice, sFRP4 mRNA and protein expression were all increased significantly in the spinal cord of ALS transgenic mice at the age of 95days, 108days, and 122days(EM>P /EM>< 0.05).The majority of sFRP4 positive cells were located in the ventral horn of the gray matter. Nerve fibers were also positive within the white matter. Compared with wild-type mice, sFRP4 positive cells were increased distinctly. sFRP4-labeled cells were co-expressed GFAP and β-tubulinIII in the ALS transgenic mice and wild-type mice, and sFRP4/GFAP double positive cells were increased in ALS transgenic mice.Conclusion BR>sFRP4 positive cells were increased significantly, and the expression of sFRP4 mRNA and protein were increased in the spinal cord of ALS transgenic mice, suggesting that sFRP4 may be closely related to the pathogenesis of amyotrophic latera