癫痫鼠外周血磷酸化S6蛋白检测及意义

Correlation of phosphorylated S6 protein expression in blood and brain tissue in mice and rats with kainic acid-induced seizure

目的:明确癫痫发作后外周血与脑组织中核糖体磷酸化S6蛋白( P-S6 )含量变化的相关性。方法:取出生后5~6周龄的C57BL/6小鼠30只和SD大鼠22只,采用红藻氨酸腹腔注射诱导癫痫发作。建立P-S6蛋白的流式细胞术检测方法用以检测小鼠及大鼠外周血中mTOR信号通路下游P-S6蛋白的变化,同时采用蛋白质印迹法检测其脑组织中P-S6的变化。采用Pearson相关性分析法分析脑组织与外周血中P-S6蛋白表达的相关性,并对大鼠外周血P-S6蛋白的表达与癫痫发作等级进行相关性分析。结果:癫痫小鼠外周血和脑组织中 P-S6 的含量明显增高,其表达量分别升高至对照组的(1.49±0.45)倍( P <0.05)和(2.55± 0.66 )倍( P <0.01);外周血中P-S6的阳性表达率和平均荧光强度均明显升高(均 P <0.01),与脑组织中P-S6蛋白表达具有一致性( r = 0.8474 , P <0.01)。大鼠自身致痫前后外周血中P-S6含量明显增加,由14.89±9.75增加至52.35±21.72( P <0.01),与大鼠脑组织P-S6蛋白表达变化一致( r =0.9385, P <0.01),且外周血P-S6含量的变化与癫痫发作等级呈正相关。结论:癫痫鼠外周血mTOR信号通路的变化与脑组织中的变化具有良好的相关性,提示通过检测外周血P-S6的表达水平可准确反映脑组织中mTOR信号通路的变化。

Objective: To determine the correlation of phosphorylated ribosomal S6 protein (P-S6) content in blood and brain tissue in mice and rats with seizure. Methods: Seizure models were induced by intraperitoric injection of kainic acid (KA) in C57BL/ mice and SD rats. Flow cytometry was used to detect the content of P-S6 in blood;Western blot was used to detect the expression of P-S6 in brain tissues. The correlation between P-S6 expression in blood and in brain tissue was examine by Pearson analysis, and the correlation between P-S6 expression in blood and the severity of seizure was also observed. Results: Western blotting analysis showed that the expression of P-S6 was significantly increased in peripheral blood and brain tissue in mice 1 h after KA-induced seizure,and the expression levels increased to (1.49± 0.45 ) times ( P <0.05) and (2.55±0.66) times ( P <0.01) of the control group, respectively. Flow cytometry showed that the positive percentage and average fluorescence intensity of P-S6 in the blood of mice increased significantly 1 h after KA-induced seizures ( P <0.01), which was consistent with the expression of P-S6 in brain tissue ( r = 0.8474 , P <0.01). Flow cytometry showed that the average fluorescence intensity of P-S6 in blood increased from 14.89±9.75 to 52.35±21.72 ( P <0.01) in rats with seizure, which was consistent with the change of P-S6 in brain tissue ( r =0.9385, P <0.01). Rats with higher levels of seizure were of higher levels of P-S6 in peripheral blood. Conclusion: Consistent correlation of P-S6 expression is demonstrated in peripheral blood and in brain tissue after KA-induced seizure, suggesting that the expression of P-S6 in blood can accurately reflect the changes of mTOR signaling pathway in brain tissue.