IL-6对小鼠胚胎心肌细胞增殖能力的影响及发育依赖性变化  

Effect of IL-6 on the Proliferative Ability of Mouse Embryonic Cardiomyocytes and Its Development-dependent Changes

作　　者：顾浩南 王淇 孙惠美[1,2] 周易 梁华敏 Gu Haonan;Wang Qi;Sun Huimei(Department of Physiology,Hubei Key Laboratory of Drug Target Research and Pharmacodynamic Evaluation,School of Basic Medicine,Huazhong University of Science and Technology,Wuhan 430030,China;National Demonstration Center for Experimental Basic Medical Education,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学基础医学院生理学系,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

出　　处：《华中科技大学学报（医学版）》2023年第6期743-748,共6页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：湖北省科技重大研发计划项目(No.2020BCB029)。

摘　　要：目的白细胞介素6(interleukin 6,IL-6)是心肌细胞损伤后心脏微环境中重要的细胞因子,可以提高心肌细胞自身的增殖能力从而促进心脏修复。本研究旨在探索IL-6对小鼠胚胎心肌细胞增殖能力的影响及其发育依赖性变化。方法用酶解法获得胚胎发育早期(early embryonic developmental stage,EDS)和发育晚期(late embryonic developmental stage,LDS)的单个心肌细胞,心肌细胞分别在20%DMEM培养液中培养24 h,然后再分别加入10 ng/mL和50 ng/mL IL-6处理24 h;用免疫荧光技术定位α-actinin^(+)心肌细胞并统计BrdU^(+)α-actinin^(+)细胞核数目/α-actinin^(+)细胞核百分比和BrdU^(+)α-actinin^(+)单核心肌细胞数目/α-actinin^(+)单核心肌细胞数目百分比,研究IL-6对心肌细胞增殖能力的影响;利用Western blot技术检测关键信号通路中关键蛋白的磷酸化水平。结果10 ng/mL IL-6不改变EDS心肌增殖能力,但对LDS心肌细胞增殖能力有显著促进作用;50 ng/mL IL-6显著抑制EDS BrdU^(+)α-actinin^(+)单核心肌细胞比例,增加LDS BrdU^(+)α-actinin^(+)细胞核和BrdU^(+)α-actinin^(+)单核心肌细胞比例。10 ng/mL IL-6和50 ng/mL IL-6抑制EDS心肌细胞Akt的磷酸化、对STAT3和ERK的磷酸化水平无显著影响;但在LDS,10 ng/mL IL-6和50 ng/mL IL-6抑制心肌细胞的Akt的磷酸化,增加STAT3的磷酸化,对ERK的磷酸化水平无显著影响。检测这3个蛋白质的基础磷酸化状态发现:STAT3、Akt和ERK的总蛋白量和磷酸化水平均呈发育依赖性下降。结论IL-6影响胚胎心肌细胞的自我更新能力,且具有发育依赖性和浓度依赖性。这可能与胚胎发育不同时期的关键蛋白及其磷酸化水平存在差异,因而IL-6在不同发育阶段靶向不同的信号通路有关。这些发现为细胞替代疗法的研究在心肌微环境层面提供了重要线索。Objective Interleukin 6(IL-6)is an important cytokine featuring pleiotropic activity in the cardiac microenvironment and improves cardiomyocyte regeneration to favor cardiac repair after injury.Thereby this study aimed to investigate the effects of IL-6 on murine embryonic cardiomyocyte proliferation and unveil the possible developmental changes.Methods Single cardiomyocyte was obtained from mouse through enzyme dissociation at the early embryonic developmental stage(EDS)and late embryonic developmental stage(LDS).The cells were cultured for 24 h in 20%DMEM and then treated with 10 ng/mL IL-6 or 50 ng/mL IL-6 for 24 h.The effect of IL-6 on cardiomyocyte proliferation was comprehensively studied by tracing BrdU^(+)α-actinin^(+)nuclei and BrdU^(+)α-actinin^(+)mononuclear cardiomyocyte with immunofluorescence staining.Western blot was performed to unveil the changes in phosphorylation levels of the key proteins under IL-6 treatments.Results The 10 ng/mL IL-6 did not change the proliferation capacity of EDS myocardium,but significantly promoted the proliferation capacity of LDS myocardium.The 50 ng/mL IL-6 significantly inhibited the proportion of EDS BrdU+α-actinin+monocytes,and increased the proportion of LDS BrdU+α-actinin+nuclei and BrdU+α-actinin+monocytes.This might be related to the various effects of IL-6 at different concentrations on signal transduction pathways of EDS and LDS cardiomyocytes:10 ng/mL IL-6 and 50 ng/mL IL-6 inhibited the phosphorylation of Akt in EDS cardiomyocytes,and had no significant effect on the phosphorylation of STAT3 and ERK.In LDS,10 ng/mL IL-6 and 50 ng/mL IL-6 inhibited phosphorylation of Akt and increased the phosphorylation of STAT3,but had no significant effect on the phosphorylation of ERK.Finally,the basic phosphorylation states of these three proteins were detected and the total protein amount and phosphorylation levels of STAT3,Akt and ERK showed a development-dependent decline.Conclusion IL-6 influenced the fetal cardiomyocyte proliferation in a developmental-and dos

关 键 词：白细胞介素6 胚胎心肌细胞 心肌再生 心脏微环境 

分 类 号：R321[医药卫生—人体解剖和组织胚胎学]