天津医药

天津医药 ›› 2017, Vol. 45 ›› Issue (2): 151-154.doi: 10.11958/20161211

• 实验研究 • 上一篇    下一篇

MeDIP-Seq 检测大鼠不同状态雪旺细胞基因甲基化水平

林伟， 樊保佑， 冯世庆△， 任一鸣， 周先虎   

  1. 天津医科大学总医院骨科 （邮编 300052）
  • 收稿日期:2016-10-24 修回日期:2016-12-16 出版日期:2017-02-15 发布日期:2017-02-14
  • 通讯作者: △通讯作者 E-mail: sqfeng@tmu.cn E-mail:Zhouxh007@126.com
  • 作者简介:林伟 （1988）， 男， 硕士研究生在读， 主要从事轴突再生方面研究
  • 基金资助:
    国家自然科学基金重点资助项目 （81330042）； 国家科技部中俄合作专项项目 （2014DFR31210）； 国家自然科学基金国际合作与交流项目 （81620108018）； 国家自然科学基金面上资助项目 （81371957）； 天津市科委重点项目 （14ZCZDSY00044， 13RCGFSY19000）

The different gene methylation levels of rat Schwann cells detected by MeDIP-Seq

LIN Wei, FAN Bao-you, FENG Shi-qing△, REN Yi-ming, ZHOU Xian-hu   

  1. Department of Orthopaedics, Tianjin Medical University General Hospital, Tianjin 300052, China
  • Received:2016-10-24 Revised:2016-12-16 Published:2017-02-15 Online:2017-02-14
  • Contact: △Corresponding Author E-mail: sqfeng@tmu.cn E-mail:Zhouxh007@126.com

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摘要： 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 7 d 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 177 176 个差异性甲基化区域， 其中位于启动子 （≤1 kb） 内 1 097 个、 启动子 （1~2 kb） 内 1 136 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。

关键词: 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

Abstract: Objective To explore the difference of DNA methylation levels between normal Schwann cells (NSCs) and activated Schwann cells (ASCs) in rats. Methods The adult Wistar rats were received sciatic nerve ligation and fed for 7 days. The ASCs and NSCs were separated from ligated sciatic nerves and brachial plexus respectively. Immunocytochemical staining of S- 100 antibody was used to identify the cells. The growth condition of cells was detected by CCK- 8 method. Methylated DNA immunoprecipitation sequencing (MeDIP-Seq) was applied to filter the differentially methylated regions in ASCs and NSCs. The distribution of differentially methylated genes related with axonal regeneration in chromosome was analyzed, and Gene ontology（GO） and PATHWAY analysis were also conducted. Results High purity of ASCs and NSCs were obtained successfully, which were both positive for S- 100 antibody. In the same culture condition, ASCs showed a faster proliferation than that of NSCs. A total of 177 176 differentially methylated regions were found by MeDIP-Seq. Among them, 1 097 were located in the promoter (≤1 kb), 1 136 in the promoter (1-2 kb) and 567 on the CpG. After functional annotation of differentially methylated genes, 214 differentially methylated genes related with axonal regeneration were found in ASCs and NSCs. Compared with NSCs, 191 genes were up-regulated and 23 genes were down -regulated in ASCs. These genes were located on different chromosomes, most of which on chromosome 12 (22 genes) and the least on chromosomes M (2 genes). GO analysis indicated that the differential methylated genes were involved in axon growth, axon formation, axon elongation and axon guidance. The MAPK, cell adhesion molecules, Ras signaling pathway may be related with the differential methylated genes. Conclusion The methylation levels between ASCs and NSCs are significantly different, which are probably related with axon regeneration.

Key words: DNA methylation, rats, Wistar, neurons, axons, computational biology, activated schwann cells, normal schwann cells, methylated DNA immunoprecipitation sequencing