癫痫持续状态后小鼠海马组织神经元中微管蛋白和内体-溶酶体系统表达的变化及其意义

doi:10.13481/j.1671-587x.20200307

吉林大学学报(医学版) ›› 2020, Vol. 46 ›› Issue (03): 470-475.doi: 10.13481/j.1671-587x.20200307

• 基础研究 • 上一篇

癫痫持续状态后小鼠海马组织神经元中微管蛋白和内体-溶酶体系统表达的变化及其意义

陈玲萌^1,2, 孟祥玥¹, 张艳³, 桂玥¹, 吴霜¹, 李艳超¹, 谭百宏⁴

1. 吉林大学基础医学院组织学与胚胎学教研室, 吉林长春 130021;
2. 吉林医药学院基础医学院人体解剖教研室, 吉林吉林 132013;
3. 吉林大学基础医学院实验教学中心, 吉林长春 130021;
4. 吉林大学生命科学公共技术中心, 吉林长春 130021

收稿日期:2019-10-15 发布日期:2020-06-11
通讯作者: 谭百宏,工程师(Tel:0431-85619477,E-mail:tanbaihong@jlu.edu.cn);李艳超,教授,博士研究生导师(Tel:0431-85619477,E-mail:liyanchao@jlu.edu.cn) E-mail:tanbaihong@jlu.edu.cn;liyanchao@jlu.edu.cn
作者简介:陈玲萌(1993-),女,吉林省德惠市人,助理实验师,医学硕士,主要从事神经元死亡机制方面的研究。
基金资助:
国家自然科学基金资助课题（81871014）

Changes of expressions of tubulin and endosome-lysosome system in neurons in hippocampus tissue of mice after status epilepticus and their significances

CHEN Lingmeng^1,2, MENG Xiangyue¹, ZHANG Yan³, GUI Yue¹, WU Shuang¹, LI Yanchao¹, TAN Baihong⁴

1. Department of Histology and Embryology, School of Basic Medical Sciences, Jilin University, Changchun 130021, China;
2. Department of Human Anatomy, School of Basic Medical Sciences, Jilin Medical College, Jilin 132013, China;
3. Core Facilities For Life Science, Jilin University, Changchun 130021, China;
4. Laboratory Teaching Center, School of Basic Medical Sciences, Jilin University, Changchun 130021, China

Received:2019-10-15 Published:2020-06-11

摘要/Abstract

摘要： 目的：探讨癫痫持续状态（SE）后小鼠海马组织神经元中微管蛋白β-tubulin和内体-溶酶体系统表达的变化，阐明神经元迟发性死亡过程中微管和内体-溶酶体系统的变化规律。方法：40只雄性ICR小鼠分为对照组（n=7，给予生理盐水）和实验组（n=33，给予匹鲁卡品），实验组中达到SE标准的SE小鼠根据SE后时间分为SE1d、SE2d、SE3d和SE7d组（n=5）。Nissl和Fluoro-Jade B （F-JB）染色检测各组小鼠海马组织神经元损伤情况，免疫荧光法检测各组小鼠海马组织神经元中微管蛋白β-tubulin、内体蛋白Rab5和溶酶体结构蛋白LAMP1表达强度及β-tubulin、Rab5和LAMP1阳性面积百分比；双重荧光法检测各组小鼠海马组织CA1区β-tubulin与Rab5和LAMP1表达的关系。结果：与对照组比较，SE1d组小鼠海马CA1和CA3区神经元数减少（P<0.01），F-JB阳性细胞数增加（P<0.01）；SE2d、SE3d和SE7d组小鼠海马组织中Nissl阳性神经元数明显减少（P<0.01）。与对照组比较，SE2d、SE3d和SE7d组小鼠海马组织中F-JB阳性神经元数增加（P<0.01）。与对照组比较，SE2d、SE3d和SE7d组小鼠海马CA1和CA3区神经元树突中β-tubulin阳性面积百分比明显降低（P<0.05），其变化趋势与神经元损伤相似。与对照组比较，SE1d、SE 2d、SE 3d和SE 7d组小鼠海马组织神经元中Rab5和LAMP1表达强度随着时间延长呈下降趋势；Rab5阳性面积百分比明显降低（P<0.05或P<0.01）；LAMP1阳性面积百分比在SE后第1天出现一过性增多，之后逐渐减少（P<0.05或P<0.01）。双重荧光染色检测，SE后1 d是早期内体减少和溶酶体一过性增多的关键点，并与神经元微管损伤的出现时间极为一致。结论：SE引起神经元迟发性死亡的同时神经元微管骨架受损，内体-溶酶体系统的定位和功能也发生异常改变。

关键词: 癫痫持续状态, 神经元, 微管蛋白, 内体-溶酶体系统, 神经元迟发性死亡

Abstract: Objective: To investigate the changes of expressions of tubulin and endosome-lysosome in the neurons in hippocampus tissue of the mice after status epilepticus (SE), and to elucidate the change rule of microtubule and endosome-lysosome system in the process of delayed neuronal death. Methods: A total of 40 male ICR mice were divided into control group(n=7,given normal saline) and experiment group(n=33, give pilocarpine);the mice in experiment group met the SE standand were divided into SE 1 d, SE 2 d, SE 3 d and SE 7 d groups according to the time after SE(n=5). Nissl and Fluoro-Jade B (F-JB) staining methods were used to detect the damage of neurons in hippocampus tissue of the mice in various groups. The expression intensities of β-tubulin,endosom protein Rab5 and lysosome constitutive protein LAMP1 and the percentages of β-tubulin, Rab5 and LAMP1 positive areas in neurons in hippocampus tissue of the mice in various groups were detected by immunofluorescence method. The relationships between the expression of β-tubulin and the expressions of Rab5 and LAMP1 in hippocampal CA1 area of the mice in various groups were detected by double fluorescence method. Results: Compared with control group, the number of neurons in hippocampal CA1 and CA3 areas of the mice in SE 1 d group was decreased (P<0.01), the number of F-JB positive cells was increased (P<0.01); the number of Nissl positive neurons in hippocampus tissue of the mice in SE 2 d, SE 3 d, and SE 7 d groups was decreased (P<0.01). Compared with control group, the number of F-JB positive neurons in hippocampus tissue of the mice in SE 2 d,SE 3 d and SE 7 d groups was increased (P<0.01). Compared with control group, the percentages of β-tubulin positive areas in hippocampal CA1 and CA3 areas of the mice in SE 2 d, SE 3 d and SE 7 d groups were significantly decreased (P<0.05), and the trend of change was similar to that of neuron damage. Compared with control group, the expression intensities of Rab5 and LAMP1 in neurons in hippocampus tissue of the mice in SE 1 d, SE 2 d, SE 3 d and SE 7 d groups were decreased with the prolongation of time, the percentages of Rab5 positive areas were significantly decreased (P<0.05 or P<0.01),and the percentages of LAMP1 positive areas were increased in a short time on the first day after SE and decreased with the prolongation of time(P<0.05 or P<0.01). The double fluorescence staining results showed that 1 d after SE was the key point for the decrease of endosome and the transient increase of lysosome in the early stage, which was consistent with the occurrence time of microtubule injury. Conclusion: SE can cause delayed neuronal death, at the same time, the tubulin skeleton of neurons is damaged, and the location and function of endosome-lysosome system are also changed abnormally.

Key words: status epilepticus, neuron, tubulin, endosomal-lysosome pathway, delayed neuronal death

中图分类号:

R361.2

陈玲萌, 孟祥玥, 张艳, 桂玥, 吴霜, 李艳超, 谭百宏. 癫痫持续状态后小鼠海马组织神经元中微管蛋白和内体-溶酶体系统表达的变化及其意义[J]. 吉林大学学报(医学版), 2020, 46(03): 470-475.

CHEN Lingmeng, MENG Xiangyue, ZHANG Yan, GUI Yue, WU Shuang, LI Yanchao, TAN Baihong. Changes of expressions of tubulin and endosome-lysosome system in neurons in hippocampus tissue of mice after status epilepticus and their significances[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 470-475.

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癫痫持续状态后小鼠海马组织神经元中微管蛋白和内体-溶酶体系统表达的变化及其意义

Changes of expressions of tubulin and endosome-lysosome system in neurons in hippocampus tissue of mice after status epilepticus and their significances

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