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中华神经创伤外科电子杂志 ›› 2018, Vol. 04 ›› Issue (01) : 33 -39. doi: 10.3877/cma.j.issn.2095-9141.2018.01.009

所属专题: 文献

基础研究

EID3参与调控人脐带间充质干细胞转分化为类神经干细胞过程的研究
陈文锦1, 罗亮1, 张丽1, 殷勤伟1, 徐如祥1,()   
  1. 1. 100700 北京,陆军总医院附属八一脑科医院
  • 收稿日期:2017-12-26 出版日期:2018-02-15
  • 通信作者: 徐如祥
  • 基金资助:
    全军"十二五"军事医学创新工程项目(BWS12J010); 中国博士后科学基金(2014M562574)

EID3 regulate the trans-differentiation process from human umbilical mesenchymal stem cell to neural stem-like cell

Wenjin Chen1, Liang Luo1, Li Zhang1, Qinwei Yin1, Ruxiang Xu1,()   

  1. 1. Affiliated BaYi Brain Hospital, PLA Army General Hospital, Beijing 100700, China
  • Received:2017-12-26 Published:2018-02-15
  • Corresponding author: Ruxiang Xu
  • About author:
    Corresponding author: Xu Ruxiang, Email:
引用本文:

陈文锦, 罗亮, 张丽, 殷勤伟, 徐如祥. EID3参与调控人脐带间充质干细胞转分化为类神经干细胞过程的研究[J]. 中华神经创伤外科电子杂志, 2018, 04(01): 33-39.

Wenjin Chen, Liang Luo, Li Zhang, Qinwei Yin, Ruxiang Xu. EID3 regulate the trans-differentiation process from human umbilical mesenchymal stem cell to neural stem-like cell[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2018, 04(01): 33-39.

目的

研究P300抑制蛋白E1A样转分化抑制蛋白3(EID3)在脐带间充质干细胞(UMSC)转分化为类神经干细胞(uNSCLs)过程中的表达变化及EID3和DNA甲基化转移酶3A(DNMT3A)之间的相互作用关系。

方法

(1)通过免疫学实验研究UMSC和uNSCLs细胞表面抗原特点;(2)通过表观遗传学基因检测、蛋白印记实验评价EID3与DNMT3A在UMSC、uNSCLs、神经干细胞(NSCs)中表达量的关系;(3)通过免疫共沉淀实验研究EID3和DNMT3A在UMSC转分化为uNSCLs过程中的相互关系。

结果

(1)uNSCLs高表达NSCs的相关mRNA,在NestinPsx6VimentinGFAPMusashi1NeuroD1基因mRNA的水平上较UMSCs增加了3~13.2倍,差异具有统计学意义(t=9.925、14.089、4.303、8.994、10.122、22.327、1.876;P=0.018、0.0005、0.026、0.0003、0.017、0.002、0.258);(2)在UMSCs转分化为uNSCLs的过程中,EID3的表达量降低,而DNMT3A则增高,DNMT3A的mRNA水平上在uNSCLs和NSCs中的表达量明显高于UMSCs,差异具有统计学意义(tuNSCLs=7.453,PuNSCLs=0.006;tNSCs=12.924,PNSCs=0.001),蛋白水平检测发现,UMSCs转分化为uNSCLs前后,EID3表达降低而DNMT3A则增高,呈相反关系;(3)在uNSCLs细胞中,EID3和DNMT3A共定位于细胞核中,在uNSCLs细胞中,EID3直接结合DNMT3A,并且,HEK293细胞内转染的外源性EID3和DNMT3A同样能相互结合。

结论

在UMSCs转分化为uNSCLs细胞的过程中,EID3的表达量逐渐增加并伴随着DNMT3A的表达量逐渐下调,同时两者发生直接结合作用,EID3参与调控了UMSC向uNSCLs的转分化过程。

Objective

To explore the expression of E1A-like inhibitor of differentiation 3 (EID3) during the trans-differentiation of umbilical cord mesenchymal stem cell (UMSC) to neural stem-like cells (uNSCLs) and the relationship between EID3 and DNA methyltransferase (DNMT3A).

Methods

(1) To explore the characteristics of UMSC and uNSCLs via immunology experiments; (2) To explore expression and correlativity of EID3 and DNMT3A in UMSC, uNSCLs and NSC via epigenetic gene screen and Western Blot; (3) To explore the co-adjustment relationship between EID3 and DNMT3A in trans-differentiation from UMSC to uNSCLs via co-immunoprecipitation assay.

Results

(1) NSCs related mRNA level are highly expressed in uNSCLs significantly, the mRNA level including Nestin, Psx6, Vimentin, GFAP, Musashi1 and NeuroD1 are 3 to 13.2 times comparing to UMSCs (t=9.925, 14.089, 4.303, 8.994, 10.122, 22.327, 1.876; P=0.018, 0.0005, 0.026, 0.0003, 0.017, 0.002, 0.258); (2) After trans-differentiation of UMSCs into uNSCLs, the expression of EID3 is down-regulated, DNMT3A is up-regulated, the DNMT3A mRNA level in uNSCLs and NSCs is highly elevated comparing to UMSCs(tuNSCLs=7.453, PuNSCLs=0.006; tNSCs=12.924, PNSCs=0.001), the protein level are the same, EID3 is declining in expression but DNMT3A is increased in expression after this trans-differentiation process; (3) EID3 and DNMT3A are co-localized in cell nucleus and EID3 interact with DNMT3A directly in uNSCLs, as well as the exogenous EID3 and DNMT3A via transfection.

Conclusion

EID3 is upregulated during trans-differentiation process from UMSCs to uNSCLs and accompany with DNMT3A down-regulated expression, EID3 may participate in this trans-differentiation process from UMSCs to uNSCLs.

图1 UMSCs和由UMSCs转分化而来的uNSCLs细胞形态及免疫学特征
图2 UMSCs、uNSCLs、NSCs中表观遗传基因mRNA水平比较及EID3和DNMT3A蛋白水平比较
图3 EID3和DNMT3A在UMSCs、uNSCLs、NSCs中免疫荧光情况
图4 EID3和DNMT3A免疫共沉淀
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