低氧通过上调内皮含PAS结构域蛋白1(EPAS1)表达促进人K562细胞向红系分化  

Hypoxia promotes erythroid differentiation of human K562 cells by upregulating endothelial PAS domain-containing protein 1(EPAS1)expression

作　　者：高瞻 肖军 李小薇 李翠莹 GAO Zhan;XIAO Jun;LI Xiaowei;LI Cuiying(Air Force Clinical College,Anhui Medical University,Beijing 100142;Department of Transfusion,Air Force Medical Center,Beijing 100142,China)

机构地区：[1]安徽医科大学空军临床学院,北京100142 [2]空军特色医学中心输血科,北京100142

出　　处：《细胞与分子免疫学杂志》2022年第6期494-500,共7页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81971778)。

摘　　要：目的探讨低氧条件下编码缺氧诱导因子2α(HIF⁃2α)的内皮含PAS结构域蛋白1(EPAS1)基因在K562人红白血病细胞向红系分化中的作用。方法将K562细胞分为常氧对照组、常氧诱导组(40μmol/L氯化血红素和0.1 ng/mL阿糖胞苷诱导)、低氧对照组(50 mL/L O_(2))、低氧诱导组。培养5 d后收集细胞,流式细胞术分析CD235a^(+)CD71^(+)细胞比例,联苯胺染色检测血红蛋白合成,CCK⁃8法检测细胞增殖,实时定量PCR检测EPAS1、胰岛素受体底物2(IRS2)、γ珠蛋白(γ⁃globin)mRNA表达,Wester blot法检测上述基因蛋白表达,分析低氧对K562细胞红系分化的影响;使用短发夹RNA(shRNA)敲低EPAS1,分析常氧及低氧下实验组(sh⁃EPAS1组)和对照组(sh⁃Control)红系分化的差异,明确EPAS1对K562细胞红系分化的作用。结果与常氧诱导组相比,低氧诱导组CD235a^(+)CD71^(+)阳性细胞显著增加,合成血红蛋白增多,EPAS1、γ⁃globin、IRS2表达明显上升,红系分化程度更高;与sh⁃Control相比,sh⁃EPAS1组CD235a^(+)CD71^(+)阳性细胞减少,合成血红蛋白降低,细胞增殖能力下降,EPAS1、γ⁃globin、IRS2表达显著降低,红系分化受阻。结论低氧能显著上调K562细胞EPAS1表达,促进细胞向红系分化。Objective To investigate the role of endothelial PAS domain⁃containing protein 1(EPAS1)gene encoded hypoxia⁃inducible factor 2α(HIF⁃2α)in erythroid differentiation of K562 human erythroleukemia cells under hypoxic conditions.Methods K562 cells were treated with 40μmol/L of hemin and 0.1 ng/mL of cytarabine for erythroid differentiation.After normoxic and hypoxic(50 mL/L O_(2))incubation,the ratio of CD235a^(+)CD71^(+) cells was detected by flow cytometry.The percentage of hemoglobin⁃positive cells was detected by benzidine staining.The level of cell proliferation was detected by CCK⁃8 assay.The mRNA and protein levels of EPAS1,insulin receptor substrate 2(IRS2)andγ⁃globin were detected by real⁃time quantitative PCR and Wester blot analysis.Besides,the changes in the erythroid differentiation of K562 cells were evaluated after knockdown of EPAS1.Results Hypoxia promoted the erythroid differentiation of K562 cells and upregulated the expression of EPAS1.After EPAS1 kncokdown,the ratio of CD235a^(+)CD71^(+) cells and hemoglobin⁃positive cells decreased,and the expressions of IRS2 andγ⁃globin declined significantly.Conclusion Hypoxia can significantly up⁃regulate the expression of EPAS1 in K562 cells and promote the erythroid differentiation.

关 键 词：低氧 内皮含PAS结构域蛋白1(EPAS1) K562细胞 红系分化 

分 类 号：R364.4[医药卫生—病理学] Q254[医药卫生—基础医学] R331.141[生物学—细胞生物学]