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心脏成纤维细胞旁分泌能力影响小鼠胚胎干细胞的命运

沈丹亚^1,2,3, 陈宗海^1,4, 赵亚楠^1,3, 周建霞^1,3, 关秀文⁵, 梁华敏^1,2,3,*

¹华中科技大学基础医学院生理学系，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华中科技大学湖北省药物靶点和药效学评价重点实验室，武汉 430030；⁴湖北民族学院病理生理学系，恩施 445000；⁵黄陂区人民医院普外科，武汉 430300

摘要

本研究旨在探讨心脏成纤维细胞旁分泌能力对小鼠胚胎干细胞的影响。新生小鼠和成年小鼠成纤维细胞的条件培养基(ConM-NCF、ConM-ACF)分别稀释50倍(1:50)或5倍(1:5)，通过RT-real time PCR技术、ELISA、细胞增殖检测以及计数心肌分化过程中跳动拟胚体(embryoid body, EB)的比例，观察这些条件培养基对小鼠胚胎干细胞的影响。结果显示，成纤维细胞细胞因子的分泌能力在发育过程中发生显著变化，其中白细胞介素6 (interleukin 6, IL6)随发育成熟显著增加。ConM-NCF抑制干细胞的增殖，但高、低浓度对干性基因表达的作用不同。ConM-ACF 1:50促进干细胞增殖并上调干性基因，但ConM-ACF 1:5作用相反。ConM-NCF和ConM-ACF都能抑制干细胞的心肌细胞分化能力，但又各有特点：ConM-NCF 1:50抑制早期心肌分化但不影响晚期心肌细胞标记物的表达；ConM-ACF 1:50抑制早期心肌分化比ConM-NCF 1:50更显著，阻碍心肌成熟并上调分化晚期的心肌细胞标记物表达水平。此外，IL6中和抗体抑制ConM-ACF 1:50对干细胞的促增殖作用，但不影响ConM-NCF 1:50的作用。干细胞分化过程中长期应用IL6中和抗体，抑制早期心肌细胞分化，但对晚期分化不产生影响。以上结果提示，心脏成纤维细胞分泌的细胞因子种类和浓度都对小鼠胚胎干细胞的增殖、干性基因表达和心肌分化有重要意义，其中ConM-ACF分泌的IL6是干细胞干性维持和心肌细胞分化的有利因素。

关键词： 小鼠胚胎干细胞; 微环境; 旁分泌; 细胞移植; 白细胞介素6

分类号：R3

Cardiac fibroblast paracrine factors modulate mouse embryonic stem cells 

SHEN Dan-Ya^1,2,3, CHEN Zong-Hai^1,4, ZHAO Ya-Nan^1,3, ZHOU Jian-Xia^1,3, GUAN Xiu-Wen⁵, LIANG Hua-Min^1,2,3,*

¹Department of Physiology, 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；³Hubei Key Laboratory of Drug Target Research and Pharmacodynamic Evaluation; Huazhong University of Science and Technology, Wuhan 430030, China；⁴Department of Pathophysiology, Hubei University of Nationalities, Enshi 445000, China；⁵Department of General Surgery, Renmin Hospital of Huangpi District, Wuhan 430300, China

Abstract

The study aims to investigate the effects of cardiac fibroblast (CF) paracrine factors on murine embryonic stem cells (ESCs). Conditioned mediums from either neonatal cardiac fibroblasts (ConM-NCF) or adult cardiac fibroblasts (ConM-ACF) were diluted by 1:50 and 1:5, respectively, to investigate whether these conditioned mediums impact murine ESCs distinctly with RT-real time PCR techniques, cell proliferation essay, ELISA and by counting percentage of beating embryoid bodies (EBs) during ESCs differentiation. The data showed that the paracrine ability of CFs changed dramatically during development, in which interleukin 6 (IL6) increased with maturation. ConM-NCF 1:50 and ConM-NCF 1:5 had opposite effects on the pluripotent markers, although they both reduced mouse ESC proliferation. ConM-ACF 1:50 promoted ESCs pluripotent markers and proliferation, while ConM-ACF 1:5 exerted negative effects. All CF-derived conditioned mediums inhibited cardiac differentiation, but with distinguishable features: ConM-NCF 1:50 slightly decreased the early cardiac differentiation without altering the maturation tendency or cardiac specific markers in EBs at differentiation of day 17; ConM-ACF 1:50 had more significant inhibitory effects on early cardiac differentiation than ConM-NCF 1:50 and impeded cardiac maturation with upregulation of cardiac specific markers. In addition, IL6 neutralization antibody attenuated positive effect of ConM-ACF 1:50 on ESCs proliferation, but had no effects on ConM-NCF 1:50. Long-term IL6 neutralization reduced the percentage of beating EBs at early developmental stage, but did not alter the late cardiac differentiation. Taken together, both the quality and quantity of factors and cytokines secreted by CFs are critical for the ESC fate. IL6 could be a favorable cytokine for ESC pluripotency and the early cardiac differentiation.

Key words: murine embryonic stem cells; microenvironment; paracrine factors; cell therapy; interleukin 6

收稿日期：2020-03-16　　录用日期：2020-08-17

通讯作者：梁华敏　　E-mail: hliang@mail.hust.edu.cn

DOI: 13294/j.aps.2020.0058

引用本文：

沈丹亚, 陈宗海, 赵亚楠, 周建霞, 关秀文, 梁华敏. 心脏成纤维细胞旁分泌能力影响小鼠胚胎干细胞的命运[J]. 生理学报 2020; 72 (5): 651-659.

SHEN Dan-Ya, CHEN Zong-Hai, ZHAO Ya-Nan, ZHOU Jian-Xia, GUAN Xiu-Wen, LIANG Hua-Min. Cardiac fibroblast paracrine factors modulate mouse embryonic stem cells . Acta Physiol Sin 2020; 72 (5): 651-659