小鼠下丘脑和海马组织神经干细胞的蛋白质组学分析

doi:10.13481/j.1671-587x.20200606

摘要/Abstract

摘要： 目的

采用蛋白质组学法探讨C57BL/6J小鼠下丘脑和海马组织中神经干细胞（NSCs）中蛋白质表达差异，阐明不同来源（区域）NSCs中蛋白表达谱不同的原因，为下丘脑和海马组织NSCs的基础研究和移植治疗提供参考。

方法

提取出生24 h内C57BL/6J小鼠原代下丘脑和海马组织 NSCs，经传代进行扩增和纯化。观察细胞形态表现，免疫荧光法检测NSCs中Nestin和Sox2的表达，采用串联质谱标签（TMT）蛋白质组学法分析下丘脑和海马组织NSCs中的差异表达蛋白质，对得到的差异蛋白质进行生物信息学分析。

结果

形态表现检测，下丘脑和海马组织NSCs均表现出不透明和折光率高的典型神经球形态。免疫荧光检测，下丘脑和海马组织NSCs中 Nestin和Sox2阳性表达率随培养代数增加而升高，在第4代达到98%以上，但下丘脑和海马组织NSCs比较差异无统计学意义（P>0.05）。蛋白质组学分析，与海马组织比较，下丘脑组织NSCs中有6种蛋白表达水平明显升高（P<0.05和P<0.01），4种蛋白表达水平明显降低（P<0.05和P<0.01）。基因本体（GO）分析，差异蛋白质主要参与包括突触传递中多巴胺摄取的负调控等8种生物过程、rRNA甲基转移酶活性等6类分子功能和高尔基体膜的组成部分等6种细胞组分。基因组百科全书（KEGG）通路分析，差异蛋白质主要参与包括囊泡运输中的SNARE相互作用等7条信号通路。

结论

体外培养的原代C57BL/6J小鼠下丘脑和海马组织NSCs的形态表现和NSCs标志物表达无差异。某些特定蛋白质表达有差异，差异表达蛋白质主要在突触传递、囊泡转运和代谢等方面发挥作用，在不同区域NSCs的定分化倾向和功能特异性方面可能起重要作用。

关键词: 下丘脑神经干细胞, 海马神经干细胞, 蛋白质组学, 差异表达蛋白质

Abstract: Objective

To explore the expression differences of proteins in the neural stem cells （NSCs） in hypothalamic tissue and hippocampal tissue in the C57BL/6J mice by proteomic analysis and clarify the reasons for different protein expression profiles of NSCs from different sources （regions）， and to provide the reference for the basic research and transplantation treatment of NSCs in hypothalamic and hippocampal tissues.

Methods

The primary NSCs were isolated from the newborn C57BL/6J mice within 24 h， and were expanded and purified by passage. The morphology of cells was observed； the expressions of Nestin and Sox2 were detected by immunofluorescence method；the differential proteins of NSCs in hypothalamic and hippocampal tissues were analyzed by tandem mass tag （TMT） proteomics， and the obtained differential proteins were analyzed by bioinformatics analysis.

Results

The morphology detection results showed that the NSCs in hypothalamic and hippocampal tissues presented the typical neurosphere morphology with opacity and high refractive index. The immunofluorescence detection results showed that the positive expression rates of Nestin and Sox2 in the NSCs in hypothalamic and hippocampal tissues were increased with the increase of culture generation， and it reached over 98% in the 4th generation， but the difference in the NSCs between hypothalamic and hippocapal tissues was not statistically significant（P>0.05）. The proteomics analysis results showed that compared with the hippocampal tissue，the expression levels of 6 kinds of proteins in the NSCs in hypothalamic tissue were significantly increased（P<0.05 or P<0.01）， while the expression levels of 4 kinds of proteins were significantly decreased（P<0.05 or P<0.01）. The gene ontology （GO） analysis results revealed that the differential proteins were mainly involved in 8 biological processes such as negative regulation of dopamine uptake involved in synaptic transmission， 6 kinds of molecular functions such as rRNA （guanine） methyltransferase activity and so on， and 6 cellular components such as integral component of Golgi membrane and so on. The Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analysis results revealed that the differential proteins were mainly involved in 7 signaling pathways such as SNARE interactions in the vesicular transport and so on.

Conclusion

The primary NSCs in hypothalamic and hippocampal tissues from C57BL/6J mice which are cultured in vitro have no differences in morphology and the expressions of NSCs markers. The expressions of certain specific proteins are different，the differentially expressed proteins mainly play a role in synaptic transmission， vesicle transport， and metabolism and may play an important role in the orientation differentiation and functional specificity of NSCs in different regions.

Key words: hypothalamic neural stem cells, hippocampal neural stem cells, proteomics, differentially expressed proteins

中图分类号:

Q189

贾雪冰,孙孟菲,姚莉,刘畅,董茵,黄日祥,柏亮,唐晨,申延琴,崔春. 小鼠下丘脑和海马组织神经干细胞的蛋白质组学分析[J]. 吉林大学学报(医学版), 2020, 46(6): 1143-1149.

Xuebing JIA,Mengfei SUN,Li YAO,Chang LIU,Yin DONG,Rixiang HUANG,Liang BAI,Chen TANG,Yanqin SHEN,Chun CUI. Proteomic analysis of neural stem cells in hypothalamic and hippocampal tissues of mice[J]. Journal of Jilin University(Medicine Edition), 2020, 46(6): 1143-1149.

图/表 4

图1

图5

表1

参考文献 21

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Accession	Protein	Change	t	P
P02802	Metallothionein-1	↑	3.547	0.024
Q64700	Retinoblastoma-like protein 2	↑	6.190	0.003
Q8BWG9	Calcium release-activated calcium channel protein 1	↑	3.383	0.028
Q91XB7	Protein YIF1A	↑	5.649	0.005
Q9QYC7	Vitamin K-dependent gamma-carboxylase	↑	3.724	0.020
Q91X88	Protein O-linked-mannose beta-1,2-N-acetylglucosaminyltransferase	↑	4.061	0.015
Q8VDS8	Syntaxin-18	↓	－6.002	0.004
Q8C0J6	Ankyrin repeat domain-containing protein SOWAHC	↓	－3.350	0.029
Q9CY21	Probable 18S rRNA (guanine-N(7))-methyltransferas	↓	－3.161	0.034
O55042	Alpha-synuclein	↓	－3.023	0.039

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