混合谱系激酶结构域样假激酶在小鼠脑缺血后脑水肿组织中的表达

Expression of mixed lineage kinase domain like pseudokinase in the edematous tissue after cerebral ischemia in mice

目的:研究混合谱系激酶结构域样假激酶(MLKL)在C57BL/6J小鼠脑缺血后脑水肿组织中的表达及作用。方法:通过向野生型(WT)和MLKL敲除(MLKL^-/^-) C57BL/6J小鼠尾静脉注射浓度为6 mg/ml玫瑰红溶液(剂量为40 mg/kg)构建缺血后脑水肿模型。一周后取组织,利用免疫荧光染色技术观察MLKL在缺血后脑水肿区的表达和分布,Western Blot检测MLKL在小鼠缺血后脑水肿区的表达水平,采用免疫电镜技术观察MLKL的亚细胞水平定位,苏木精-伊红染色(HE)观察组织缺血坏死及水肿情况。结果:免疫荧光染色结果显示正常WT小鼠大脑皮质无MLKL表达,缺血后脑损伤区的WT小鼠有大量MLKL阳性细胞,MLKL主要表达于GFAP阳性星形胶质细胞; Western Blot结果显示脑缺血后脑损伤区的MLKL表达显著增加;免疫电镜结果显示脑水肿区域血管周围的星形胶质细胞内有大量MLKL蛋白胶体金颗粒分布; HE染色结果显示MLKL敲除可显著减轻脑水肿程度。结论:脑缺血可上调WT小鼠缺血部位MLKL表达,提示MLKL可能参与缺血后的脑水肿发生。

Objective: To investigate the expression and possible roles of mixed lineage kinase domain like pseudokinase( MLKL) in ischemia-induced edema. Methods: Focal cerebral ischemia is made by photothrombosis. Brain tissues were collected at 1 week after ischemia. Immunohistochemistry,Western Blot,immune-electron microscopic study and HE staining were conducted to explore the expression of MLKL in ischemia-induced edema. Results: Immunohistochemistry showed that there was no expression of MLKL in normal cortex. Ischemia induces expression of MLKL in injured cortex,mainly in GFAP-positive astrocytes. Western Blot confirmed the up-regulation of MLKL. Immuno-electron microscopic results showed that MLKL-immuonreactivity specifically localized in the cytoplasm of astrocytes which surround the blood vessel. HE staining showed that knocking out MLKL could significantly reduce the ischemia-induced edema. Conclusion: MLKL is upregualted in edema area of ischemic cortex in mice. Knocking out MLKL could alleviate the ischemia-induced edema.