摘要/Abstract

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
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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