上海交通大学学报（医学版）

上海交通大学学报（医学版） >

2019 , Vol. 39 >Issue 1: 4

DOI: https://doi.org/10.3969/j.issn.1674-8115.2019.01.002

论著·基础研究

多能干细胞关键因子Oct4 RNA结合蛋白的分离与鉴定

  • 马姬 1* ,
  • 郭传亮 2 ,
  • 3* ,
  • 范书玥 1 ,
  • 3 ,
  • 薛燕 1 ,
  • 2 ,
  • 3 ,
  • 曾凡一 1 ,
  • 2 ,
  • 3
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  • 1. 上海交通大学基础医学院组织胚胎学与遗传发育学系，上海 200025；2. 上海交通大学附属儿童医院上海医学遗传研究所，上海 200040； 3. 上海市胚胎与生殖工程重点实验室，国家卫生和健康委员会医学胚胎分子生物学重点实验室，上海 200040
马姬（1973—），女，实验师，硕士生；电子信箱：maanshan0555@sina.com。郭传亮（1986—），男，助理研究员，博士；电子信箱：331364650@qq.com。*为共同第一作者。

网络出版日期: 2019-02-27

基金资助

国家重点科学研究计划（ 2014CB964701，2014CB964703）；国家自然科学基金（ 31871484）；上海市“重中之重”临床医学中心和临床重点学科建设项目（ 2017ZZ02019）；上海市自然科学基金（ 16ZR1428600）

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Isolation and identification ofOct4 RNA-binding proteins

  • MA Ji1* ,
  • GUO Chuan-liang2 ,
  • 3* ,
  • FAN Shu-yue1 ,
  • 3 ,
  • XUE Yan1 ,
  • 2 ,
  • 3 ,
  • ZENG Fan-yi1 ,
  • 2 ,
  • 3
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  • 1. Department of Histoembryology, Genetics & Development, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China; 2. Shanghai Institute of Medical Genetics, Childrens Hospital of Shanghai Jiao Tong University, Shanghai 200040, China; 3. Shanghai Key Laboratory of Embryo and Reproduction Engineering, National Health Commission of the Peoples Republic of China Key Laboratory of Medical Embryogenesis and Developmental Molecular Biology, Shanghai 200040, China

Online published: 2019-02-27

Supported by

China National Basic Research Program, 2014CB964701, 2014CB964703; National Natural Science Foundation of China, 31871484; Constructing Project of Clinical Medical Centers and Clinical Key Disciplines of Shanghai, 2017ZZ02019; Natural Science Foundation of Shanghai, 16ZR1428600

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摘要

目的 ·分离多能干细胞关键因子 Oct4的 RNA结合蛋白。方法 ·培养小鼠胚胎干细胞（ R1），从中提取细胞总 RNA，反转录为 cDNA，PCR扩增 Oct4并转录为 mRNA。采用链霉素亲和层析和质谱技术筛选候选 RNA结合蛋白，采用 RNA免疫沉淀技术鉴定候选 RNA结合蛋白。结果 ·经高效液相色谱 -质谱法鉴定和筛选出 121个能与 Oct4 3非翻译区（ untranslated region，UTR）和 Oct4 5 UTR相结合的 RNA结合蛋白，肽段图谱匹配数≥ 2的有 11个。并对其中的 7个候选 RNA结合蛋白进行了进一步验证，结果显示 Oct4可与 DSP、SOD1、LMNA、NPM1、PSIP1、NCL和 HDGF蛋白发生相互作用，其中 HDGF与 Oct4结合能力最强。结论 · Oct4 RNA结合蛋白的鉴定将为 Oct4的调控机制提供一定理论依据，为后续其功能研究提供基础。

关键词： Oct4; RNA结合蛋白; 胚胎干细胞; RNA免疫沉淀

本文引用格式

马姬 1* , 郭传亮 2 , 3* , 范书玥 1 , 3 , 薛燕 1 , 2 , 3 , 曾凡一 1 , 2 , 3 . 多能干细胞关键因子Oct4 RNA结合蛋白的分离与鉴定[J]. 上海交通大学学报（医学版）, 2019 , 39(1) : 4 . DOI: 10.3969/j.issn.1674-8115.2019.01.002

Abstract

Objective · To identify the RNA binding proteins of Oct4, a key factor of embryonic stem cells. Methods · Total RNA was extracted moembryonic stem cells (R1), which was reverse-transcribed into cDNA. Then PCR product of Oct4 mRNA were transcribed into Oct4 mRNA. Finally the candidate RNA-binding proteins were eluted applying the streptomycin affinity chromatography, and submitted for high-performance liquid chromatography combined with the mass spectrometry. The identified RNA binding proteins were further confirmedRNA immunoprecipitation (RIP). Results · 121 RNA binding proteins of Oct4 3 untranslated region (UTR) and Oct4 5 UTR were identified with liquid chromatography / mass spectrometry. There were 11 proteins with the number of peptide spectrum matches more or equal to 2, and 7 of them were selected for additional confirmation using RIP method. RIP results showed that Oct4 interacted with DSP, SOD1, LMNA, NPM1, PSIP1, NCL and HDGF. Among them, HDGF had the strongest binding ability to Oct4 mRNA. Conclusion · Identification of Oct4 RNA binding proteins will provide a theoretical basis for the regulation mechanism of Oct4, and will be a basis for the further study of its function.

Key words： Oct4; RNA binding protein; embryonic stem cell; RNA immunoprecipitation

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