MeDIP-Seq检测大鼠不同状态雪旺细胞基因甲基化水平

目的探寻大鼠正常态雪旺细胞（NSCs）和激活态雪旺细胞（ASCs）差异甲基化基因。方法成年Wistar大鼠结扎坐骨神经，饲养7d后分别从坐骨神经和臂丛神经提取ASCs和NSCs。S-100抗体免疫荧光染色鉴定细胞，CCK-8法测定细胞生长情况。甲基化免疫共沉淀测序（MeDIP-Seq）技术筛选出ASCs和NSCs的差异性甲基化区域；分析与轴突再生相关的差异甲基化基因在染色体的分布情况，并进行基因本体（GO）和信号通路（PATHWAY）分析。结果成功获得了高纯度的ASCs和NSCs，两者S-100均表达阳性。在相同培养条件下，ASCs生长速度更快。MeDIP-Seq共发现177176个差异性甲基...

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Bibliographic Details
Published in天津医药 Vol. 45; no. 2; pp. 151 - 154
Main Author 林伟;樊保佑;冯世庆;任一鸣;周先虎
Format Journal Article
LanguageChinese
Published 天津医科大学总医院骨科 邮编300052 2017
Subjects
DNA甲基化;大鼠,Wistar;神经元;轴突;计算生物学;激活态雪旺细胞;正常态雪旺细胞;甲基化免疫共沉淀测序
激活态雪旺细胞
computational biology
DNA甲基化
normal schwann cells
神经元
neurons
activated schwann cells
axons
甲基化免疫共沉淀测序
计算生物学
DNA methylation
rats,Wistar
methylated DNA immunoprecipitation sequencing
大鼠,Wistar
正常态雪旺细胞
轴突
Online AccessGet full text
ISSN0253-9896
DOI10.11958/20161211

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Summary:目的探寻大鼠正常态雪旺细胞（NSCs）和激活态雪旺细胞（ASCs）差异甲基化基因。方法成年Wistar大鼠结扎坐骨神经，饲养7d后分别从坐骨神经和臂丛神经提取ASCs和NSCs。S-100抗体免疫荧光染色鉴定细胞，CCK-8法测定细胞生长情况。甲基化免疫共沉淀测序（MeDIP-Seq）技术筛选出ASCs和NSCs的差异性甲基化区域；分析与轴突再生相关的差异甲基化基因在染色体的分布情况，并进行基因本体（GO）和信号通路（PATHWAY）分析。结果成功获得了高纯度的ASCs和NSCs，两者S-100均表达阳性。在相同培养条件下，ASCs生长速度更快。MeDIP-Seq共发现177176个差异性甲基化区域，其中位于启动子（≤1kb）内1097个、启动子（1~2kb）内1136个，CpG岛内567个。共获得214个与轴突再生相关的差异甲基化基因，与NSCs相比，ASCs高甲基化基因191个，低甲基化基因23个。这些基因位于各个染色体上，以12号染色体上最多（22个），M染色体最少（2个）。GO分析结果显示差异甲基化基因涉及了轴突生长、轴突形成、轴突延伸和轴突导向等过程；并且与MAPK、细胞黏附分子和Ras等信号通路有关。结论ASCs和NSCs的甲基化水平存在明显的差异，可能与轴突再生有关。
Bibliography:Objective To explore the difference of DNA methylation levels between normal Schwann cells(NSCs)andactivated Schwann cells(ASCs)in rats.Methods The adult Wistar rats were received sciatic nerve ligation and fed for7days.The ASCs and NSCs were separated from ligated sciatic nerves and brachial plexus respectively.Immunocytochemicalstaining of S-100antibody was used to identify the cells.The growth condition of cells was detected by CCK-8method.Methylated DNA immunoprecipitation sequencing(MeDIP-Seq)was applied to filter the differentially methylated regions inASCs and NSCs.The distribution of differentially methylated genes related with axonal regeneration in chromosome wasanalyzed,and Gene ontology(GO)and PATHWAY analysis were also conducted.Results High purity of ASCs and NSCswere obtained successfully,which were both positive for S-100antibody.In the same culture condition,ASCs showed afaster proliferation than that of NSCs.A total of177176differentially methylated regions were found by MeDIP-Seq.Amongthem,
ISSN:0253-9896
DOI:10.11958/20161211