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浙江大学学报（医学版）
浙江大学学报（医学版）  2019, Vol. 48 Issue (3): 303-309    DOI: 10.3785/j.issn.1008-9292.2019.06.11
原著     
癫痫鼠外周血磷酸化S6蛋白检测及意义
范苗1(),董淑敏1,邹心怡1,郑博远1,黄玉润1,王健达2,*(),曾玲晖1,*()
1. 浙江大学城市学院医学院, 浙江 杭州 310015
2. 浙江大学医学院附属第二医院儿科, 浙江 杭州 310009
Correlation of phosphorylated S6 protein expression in blood and brain tissue in mice and rats with kainic acid-induced seizure
FAN Miao1(),DONG Shuminin1,ZOU Xinyi1,ZHENG Boyuan1,HUANG Yurun1,WANG Jianda2,*(),ZENG Linghui1,*()
1. School of Medicine, Zhejiang University City College, Hangzhou 310015, China
2. Department of Paediatrics, the Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou 310009, China
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摘要：

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。
关键词： 癫痫/病理学;  癫痫/血液;  脑/解剖学和组织学;  蛋白激酶类/生理学;  磷蛋白磷酸酶类/代谢;  流式细胞术;  疾病模型, 动物    
Abstract:

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.
Key words: Epilepsy/pathology    Epilepsy/blood    Brain/anatomy &    histology    Protein kinases/physiology    Phosphoprotein phosphatases/metabolism    Flow cytometry    Disease models, animal
收稿日期: 2019-01-30 出版日期: 2019-09-04
CLC:  R421.1  
基金资助: 杭州市重大科技创新项目(20152013A02);国家自然科学基金(81600988)
通讯作者: 王健达,曾玲晖     E-mail: 15958027903@163.com;2515165@zju.edu.cn;zenglh@zucc.edu.cn
作者简介: 范苗(1995-), 女, 硕士研究生, 主要从事神经损伤性疾病机制及药物治疗研究; E-mail: 15958027903@163.com; https://orcid.org/0000-0002-3220-6762
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范苗
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曾玲晖

引用本文:

范苗, 董淑敏, 邹心怡, 郑博远, 黄玉润, 王健达, 曾玲晖. 癫痫鼠外周血磷酸化S6蛋白检测及意义[J]. 浙江大学学报（医学版）, 2019, 48(3): 303-309.

FAN Miao, DONG Shuminin, ZOU Xinyi, ZHENG Boyuan, HUANG Yurun, WANG Jianda, ZENG Linghui. Correlation of phosphorylated S6 protein expression in blood and brain tissue in mice and rats with kainic acid-induced seizure. J Zhejiang Univ (Med Sci), 2019, 48(3): 303-309.

链接本文:

http://www.zjujournals.com/med/CN/10.3785/j.issn.1008-9292.2019.06.11    或    http://www.zjujournals.com/med/CN/Y2019/V48/I3/303

图 1  癫痫小鼠外周血和脑组织中磷酸化S6蛋白电泳条带(n=6)
图 2  癫痫小鼠外周血磷酸化S6蛋白流式细胞术检测结果(n=12)
图 3  癫痫小鼠脑组织与外周血中磷酸化S6蛋白表达水平的相关性
图 4  癫痫大鼠外周血和脑组织中磷酸化S6蛋白含量变化(n=10)
图 5  癫痫大鼠脑组织与外周血中磷酸化S6蛋白表达水平的相关性
图 6  癫痫大鼠不同癫痫发作等级外周血磷酸化S6蛋白含量的变化(n=16)
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