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IL-6促进小鼠胚胎干细胞多能性并以发育依赖的方式调控心肌分化

季思琦^1,2, 赵亚楠^1,2, 周建霞^1,2, 陈宗海^1,2,3, 梁华敏^1,2,*

¹华中科技大学基础医学院生理学系，中德干细胞中心，湖北省药物靶点和药效学评价重点实验室，武汉 430030；²华中科技大学基础医学国家级实验教学示范中心，武汉 430030；³湖北民族学院病理生理学系，恩施 445000

摘要

白细胞介素6 (interleukin 6, IL-6)是心脏微环境的重要组成部分。在不同模型中，IL-6通过促进心肌细胞再生帮助心脏修复。胚胎干细胞是心脏修复中心肌再生的良好来源。本研究旨在探讨IL-6对小鼠胚胎干细胞(mouse embryonic stem cells, mESCs)及其心肌分化的影响。IL-6处理mESCs两天，用CCK-8法检测mESCs增殖情况，用实时荧光定量PCR (quantitative real-time PCR, qPCR)检测干性和胚层分化基因mRNA表达水平，用Western blot检测干细胞相关信号通路的磷酸化水平，用siRNA干扰STAT3磷酸化功能。通过检测搏动拟胚体(embryoid bodies, EBs)比例和qPCR检测心脏前体细胞标记物和心肌细胞离子通道mRNA表达水平来评估mESCs的分化能力。从胚胎干细胞分化第1天(EB0)开始使用IL-6中和抗体阻断内源性IL-6的作用，分别在EB7、EB10和EB15检测心肌细胞分化；在EB15用免疫组织化学染色法示踪心肌细胞，并用Western blot检测干细胞相关信号通路的磷酸化情况。在EB4、EB7、EB10和EB15时给予短期IL-6抗体处理，观察发育后期搏动EBs比例。结果显示，外源性IL-6促进mESCs增殖和多能性的维持，表现为上调癌基因c-fos、c-jun和干性标志物oct4、nanog mRNA表达水平，下调胚层分化标记物branchyury、FLK-1、pecam、ncam和sox17 mRNA表达水平，提高ERK1/2和STAT3磷酸化水平；针对JAK/STAT3的siRNA可部分减弱IL-6对细胞增殖和c-fos、c-jun mRNA表达水平的作用。在分化过程中，IL-6中和抗体的长期作用可减小搏动EBs比例，下调ISL1、GATA4、α-MHC、cTnT、kir2.1和cav1.2 mRNA水平，降低EBs和单个细胞的心肌α actinin荧光强度，下调STAT3磷酸化水平。此外，从EB4开始的短期(2 d) IL-6抗体处理显著降低发育后期搏动EBs的比例，而从EB10开始的短期IL-6抗体处理显著提高EB16搏动EBs的比例。以上结果提示，外源性IL-6促进mESCs增殖，有利于干细胞干性维持；内源性IL-6对mESCs心肌分化的影响呈发育依赖性。这些发现为细胞替代疗法的微环境研究提供了重要依据，也为理解心脏疾病的病理生理学提供了新的视角。

关键词： 胚胎干细胞; 心肌分化; 白细胞介素6; 心肌再生

IL-6 promotes pluripotency of mouse embryonic stem cells and regulates cardiac differentiation in a development-dependent manner

JI Si-Qi^1,2, ZHAO Ya-Nan^1,2, ZHOU Jian-Xia^1,2, CHEN Zong-Hai^1,2,3, LIANG Hua-Min^1,2,*

¹Department of Physiology, Chinese-German Stem Cell Center, Hubei Key Laboratory of Drug Target Research and Pharmacodynamic Evaluation (Huazhong University of Science and Technology), School of Basic Medicine, Huazhong University of Science and Technology, Wuhan 430030, China；²National Demonstration Center for Experimental Basic Medical Education, Huazhong University of Science and Technology, Wuhan 430030, China；³Department of Pathophysiology, Hubei University of Nationalities, Enshi 445000, China

Abstract

Interleukin 6 (IL-6), an important component of cardiac microenvironment, favors cardiac repair by improving cardiomyocyte regeneration in different models. This study aimed to investigate the effects of IL-6 on stemness maintenances and cardiac differentiation of mouse embryonic stem cells (mESCs). The mESCs were treated with IL-6 for two days, and then subjected to CCK-8 essay for proliferation analysis and quantitative real-time PCR (qPCR) to evaluate the mRNA expression of genes related to stemness and germinal layers differentiation. Phosphorylation levels of stem cell-related signal pathways were detected by Western blot. siRNA was used to interfere the function of STAT3 phosphorylation. Cardiac differentiation was investigated by the percentage of beating embryoid bodies (EBs) and qPCR analysis of cardiac progenitor markers and cardiac ion channels. IL-6 neutralization antibody was applied to block the endogenous IL-6 effects since the onset of cardiac differentiation (embryonic day of 0, EB0). The EBs were collected on EB7, EB10 and EB15 to investigate the cardiac differentiation by qPCR. On EB15, Western blot was applied to investigate the phosphorylation of several signaling pathways, and immunochemistry staining was adopted to trace the cardiomyocytes. IL-6 antibody was administered for two days (short term) on EB4, EB7, EB10 or EB15, and percentages of beating EBs at late developmental stage were recorded. The results showed that exogenous IL-6 promoted mESCs proliferation and favored maintenances of pluripotency, evidenced by up-regulated mRNA expression of oncogenes (c-fos, c-jun) and stemness markers (oct4, nanog), down-regulated mRNA expression of germ layer genes (branchyury, FLK-1, pecam, ncam, sox17), and increased phosphorylation of ERK1/2 and STAT3. siRNA targeting JAK/STAT3 partially attenuated the effects of IL-6 on cell proliferation and mRNA expression of c-fos and c-jun. During differentiation, long term IL-6 neutralization antibody application decreased the percentage of beating EBs, down-regulated mRNA expression of ISL1, GATA4, α-MHC, cTnT, kir2.1, cav1.2, and declined the fluorescence intensity of cardiac α actinin in EBs and single cell. Long term IL-6 antibody treatment decreased the phosphorylation of STAT3. In addition, short term (2 d) IL-6 antibody treatment starting from EB4 significantly reduced the percentage of beating EBs in late development stage, while short term IL-6 antibody treatment starting from EB10 significantly increased the percentage of beating EBs on EB16. These results suggest that exogenous IL-6 promotes mESCs proliferation and favors stemness maintenance. Endogenous IL-6 regulates mESC cardiac differentiation in a development-dependent manner. These findings provide important basis for the study of microenvironment on cell replacement therapy, as well as a new perspective for understanding the pathophysiology of heart diseases.

Key words: embryonic stem cells; cardiac differentiation; interleukin 6; cardiomyocyte regeneration

收稿日期：　　录用日期：

通讯作者：梁华敏　　E-mail:

DOI: 10.13294/j.aps.2023.0010

引用本文：

季思琦, 赵亚楠, 周建霞, 陈宗海, 梁华敏. IL-6促进小鼠胚胎干细胞多能性并以发育依赖的方式调控心肌分化[J]. 生理学报 2023; 75 (1): 49-58.

JI Si-Qi, ZHAO Ya-Nan, ZHOU Jian-Xia, CHEN Zong-Hai, LIANG Hua-Min. IL-6 promotes pluripotency of mouse embryonic stem cells and regulates cardiac differentiation in a development-dependent manner. Acta Physiol Sin 2023; 75 (1): 49-58