RhoA和ROCK2在SOD1-G93A转基因小鼠脊髓中的表达

The expression of RhoA and ROCK2 in the spinal cord of SOD1-G93A transgenic mice

目的:通过研究RhoA和ROCK2在SOD1-G93A转基因小鼠脊髓内的表达变化以阐明Rho/ROCK信号通路在肌萎缩侧索硬化症(ALS)病程中的作用。方法:饲养SOD1-G93A转基因小鼠和同窝野生型小鼠至发病早期、中期和晚期,部分小鼠冰上剥离新鲜脊髓组织,利用RT-PCR方法检测RhoA和ROCK2 mRNA的表达,利用Western Blot方法检测RhoA和ROCK2蛋白的表达;部分小鼠行心脏灌注并剥离其脊髓组织制成冰冻切片,利用免疫组织化学染色方法检测RhoA和ROCK2蛋白的表达。结果:在SOD1-G93A鼠发病的早期、中期和晚期,转基因小鼠脊髓中RhoA和ROCK2的mRNA及蛋白表达均上调。免疫组织化学染色实验结果显示,野生型小鼠脊髓中RhoA和ROCK2弥散分布于胞质和突起中,阳性染色浅,SOD1-G93A转基因小鼠脊髓中RhoA和ROCK2阳性染色深,大量聚集在细胞膜及细胞质。结论:RhoA和ROCK2在SOD1-G93A转基因小鼠脊髓中异常高水平表达与ALS脊髓区病变密切相关,可能参与ALS疾病进程。

Objective:To elucidate the role of Rho/ROCK signaling pathway in the progression of ALS by detecting the changes of RhoA and ROCK2 expression in the spinal cord of SOD1-G93A transgenic mice.Methods:SOD1-G93A transgenic mice and littermate wild-type mice were raised to the early,middle and late stages of the disease.A part of mice were killed to dissect fresh spinal cord tissue on ice for RT-PCR and Western Blot experiments.Another part of mice were perfused intracardially and fixed,then the spinal cord tissues were separated to prepare frozen sections for immunohistochemical staining.Results:RT-PCR and Western Blot assays showed that in the early,middle and late stages of the disease,up-regulated mRNA and protein expressions of RhoA and ROCK2 in the spinal cord of SOD1-G93A transgenic mice were found compared with wide-type mice.The results of immunohistochemical staining showed that RhoA and ROCK2 were lightly stained and diffusely distributed in the cytoplasm and processes of the spinal cord of wildtype mice,while RhoA and ROCK2 were deeply positive stained in SOD1-G93A transgenic mice,and a large amount of RhoA and ROCK2 were clustered in the cell membrane and cytoplasm.Conclusion:The abnormally high expressions of RhoA and ROCK2 in the spinal cord of SOD1-G93A transgenic mice were closely related to the lesions of the ALS spinal cord and might be involved in the progression of ALS.