MeDIP-Seq检测大鼠不同状态雪旺细胞基因甲基化水平  

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

作　　者：林伟[1] 樊保佑 冯世庆[1] 任一鸣[1] 周先虎[1] LIN Wei;FAN Bao-you;FENG Shi-qing;REN Yi-ming;ZHOU Xian-hu(Department of Orthopaedics, Tianjin Medical University General Hospital, Tianjin 300052, China)

机构地区：[1]天津医科大学总医院骨科,300052

出　　处：《天津医药》2017年第2期151-154,共4页Tianjin Medical Journal

基　　金：国家自然科学基金重点资助项目(81330042);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);国家科技部中俄合作专项项目(2014DFR31210);天津市科委重点项目(14ZCZDSY00044;13RCGFSY19000)

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(Me DIP-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。Me DIP-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,Cp G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。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,1097were located in the promoter(≤1kb),1136in the promoter(1-2kb)and567on the CpG.After functionalannotation of differentially methylated genes,214differentially methylated genes related with axonal regeneration were foundin ASCs and NSCs.Compared with NSCs,191genes were up-regulated and23genes were down-regulated in ASCs.Thesegenes were located on different chromosomes,most of which on chromosome12(22genes)and the least on chromosomes M(2genes).GO analysis indicated that the differential methylated genes were involved in axon growth,axon formation,axonelongation and axon guidance.The MAPK,cell adhesion molecules,Ras signaling pathway may be related with thedifferential methylated genes.Conclusion The methylation levels between ASCs and NSCs are significantly different,which are probably related with axon regeneration.

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 

分 类 号：R338.1[医药卫生—人体生理学]