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目的检测Wnt抑制因子-1(Wif-1)在成年肌萎缩脊髓侧索硬化症(ALS)转基因模型小鼠脊髓内的表达变化,进一步探讨Wif-1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54只,分别于鼠龄95d、108d、122d取材,部分鼠经4%多聚甲醛心脏灌注固定、分离脊髓、制备冷冻切片,应用免疫荧光染色技术检测脊髓内Wif-1的分布及变化;部分鼠取出脊髓、匀浆,应用RT-PCR和Western blotting方法观察不同时间点Wif-1 mRNA及其蛋白表达水平变化。结果成年ALS转基因鼠和同窝野生型鼠脊髓的中央管、灰质、白质中均可检测到Wif-1阳性细胞,在灰质内,Wif-1阳性细胞主要分布于脊髓前角;在白质内,神经元的轴突呈阳性反应。与同窝野生型鼠比较,ALS转基因鼠Wif-1阳性细胞显著增多,Wif-1 mRNA和蛋白表达在95d变化不明显,在108d、122d均有升高,差异具有统计学意义(P<0.05),其中108d升高最明显。结论在ALS转基因鼠发病过程中Wif-1阳性细胞明显增多,Wif-1 mRNA和蛋白表达升高,表明Wif-1与ALS的发生密切相关。
Objective To detect the expression of Wnt inhibitor-1 (Wif-1) in the spinal cord of adult mouse model with ALS and to further explore the role of Wif-1 in the pathogenesis of ALS. Methods Fifty-four ALS transgenic mice and wild-type littermates were selected and harvested at 95d, 108d and 122d respectively. Some of the mice were perfused with 4% paraformaldehyde via cardiac perfusion. The spinal cord was isolated and frozen sections were prepared. Immunofluorescence staining The distribution and changes of Wif-1 in the spinal cord were detected. Some rats were removed the spinal cord and homogenized. The changes of Wif-1 mRNA and protein expression at different time points were observed by RT-PCR and Western blotting. Results Wif-1 positive cells could be detected in the central tube, gray matter and white matter of adult ALS transgenic mice and wild-type littermates. Within the gray matter, Wif-1 positive cells were mainly distributed in the anterior horn of the spinal cord. In the white matter, Neurons axon positive reaction. Compared with wild-type littermates, the Wif-1 positive cells in ALS transgenic mice increased significantly, while the expression of Wif-1 mRNA and protein did not change significantly at 95d, and increased at 108d and 122d (P <0.05) ), The most obvious increase in 108d. Conclusions Wif-1 positive cells increased significantly and the expression of Wif-1 mRNA and protein increased during the pathogenesis of ALS transgenic mice, indicating that Wif-1 is closely related to the occurrence of ALS.