Impact of Coxsackievirus B3 Infection on HL-1 Cardiomyocytes and Mesenchymal Stem Cells

doi:10.12114/j.issn.1007-9572.2018.00.249

Abstract

Abstract: Background　Dilated cardiomyopathy（DCM）is characterized by dilated heart chambers and decreased contractile function，with or without heart failure.Myocarditis contributes to dilated cardiomyopathy，and Coxsackie Virus B（CVB）is the commonest virus which could lead to myocarditis.It was widely reported that mesenchymal stem cells（MSCs）applied for acute myocardial infarction and heart failure，but seldom used for the research of myocarditis.Objective　To compare the impact of Coxsackievirus B3（CVB3）infection on HL-1 cardiomyocytes and mesenchymal stem cells in order to find a new treatment for myocarditis.Methods　We carried out this study from June 2015 to June 2017.HL-1 cardiomyocytes and MSCs were cultivated separately to 80% confluence for use.HL-1 cardiomyocytes and MSCs were divided into non-CVB3-infected group（cultured in serum free medium for 1 hour），and CVB3-infected groups〔including 4-hour-infection group（infected with CVB3 for 4 hours），12-hour-infection group（infected with CVB3 for 12 hours），24-hour-infection group（infected with CVB3 for 24 hours）〕，respectively.qPCR was used to detect CVB3 genome copy numbers in infected groups of HL-1 cardiomyocytes and MSCs.HL-1 cardiomyocytes and MSCs were divided into non-CVB3-infected group（cultured in serum free medium for 1 hour），and CVB3-infected group（infected with CVB3）.MTS cell proliferation assay was adopted to determine the cell viability of non-CVB3-infected group and CVB3-infected group of HL-1 cardiomyocytes and MSCs at 4，12，24 h after infection.CVB3 was used to infect HL-1 cardiomyocytes（CVB3-infected HL-1 cardiomyocytes group）and MSCs（CVB3-infected MSCs group）.Plaque assay was employed to measure the viral titers in CVB3-infected groups of HL-1 cardiomyocytes and MSCs.Results　The CVB3 genome copy number differed significantly between non-CVB3-infected HL-1 cardiomyocytes group，4-hour-CVB3-infected HL-1 cardiomyocytes group，12-hour-CVB3-infected HL-1 cardiomyocytes group，and 24-hour-CVB3-infected HL-1 cardiomyocytes group（P<0.05）．Similarly，it varied obviously between non-CVB3-infected MSCs group，4-hour-CVB3-infected MSCs group，12-hour-CVB3-infected MSCs group，and 24-hour-CVB3-infected MSCs group（P<0.05）．The cell viability at 4，12，24 h after infection of non-CVB3-infected HL-1 cardiomyocytes group was much higher than that of the CVB3-infected HL-1 cardiomyocytes group（P<0.05）．The cell viability in CVB3-infected HL-1 cardiomyocytes group varied significantly at different time points（P<0.05），but that of the non-CVB3-infected HL-1 cardiomyocytes group did not（P>0.05）．Non-CVB3-infected MSCs group and CVB3-infected MSCs group showed similar cell viability（P>0.05）．The cell viability of non-CVB3-infected MSCs group and CVB3-infected MSCs group changed significantly at different time points（P<0.05）．The viral titer in CVB3-infected HL-1 cardiomyocytes group was much higher than that of CVB3-infected MSCs group（P<0.05）．Conclusion　CVB3-infected HL-1 cardiomyocytes showed decreased cell viability and replication of CVB3，but CVB3-infected MSCs presented insignificantly changed cell viability and non-replication of CVB3.

Key words: Coxsackievirus infections；Enterovirus B, human；Myocytes, cardiac；Mesenchymal stem cells

摘要： 背景　扩张型心肌病（DCM）是一种以心腔扩大及心肌收缩功能减退，伴或不伴充血性心力衰竭的心肌病。病毒性心肌炎是形成DCM的重要病因，而引起心肌炎的病毒以柯萨奇B组病毒（CVB）最为常见。间充质干细胞（MSC）已广泛应用于急性心肌梗死及慢性心力衰竭的实验研究中，但其应用于心肌炎的研究较少。目的　比较柯萨奇B3病毒（CVB3）感染对HL-1心肌细胞及MSC的影响，以期寻找一种新的心肌炎的治疗措施。方法　2015年6月—2017年6月，分别培养HL-1心肌细胞、MSC，生长至80%汇合状态待用。分别将HL-1心肌细胞、MSC分为未感染组、感染后4 h组、感染后12 h组、感染后24 h组，其中感染后4 h组、感染后12 h组、感染后24 h组分别用CVB3感染4、12、24 h，未感染组仅用无血清培养基培养1 h，采用实时荧光定量PCR法检测CVB3基因拷贝数。分别将HL-1心肌细胞、MSC分为未感染组、感染组，其中感染组用CVB3感染，未感染组仅用无血清培养基培养1 h，分别于感染后4、12、24 h采用MTS法检测细胞活性。用CVB3分别感染HL-1心肌细胞（HL-1心肌细胞组）、MSC（MSC组），采用病毒空斑实验检测病毒滴度。结果　HL-1心肌细胞未感染组、感染后4 h组、感染后12 h组、感染后24 h组CVB3基因拷贝数比较，差异有统计学意义（P<0.05）。MSC未感染组、感染后4 h组、感染后12 h组、感染后24 h组CVB3基因拷贝数比较，差异有统计学意义（P<0.05）。HL-1心肌细胞感染组感染后4、12、24 h细胞活性小于HL-1心肌细胞未感染组（P<0.05）。HL-1心肌细胞未感染组不同时间点细胞活性比较，差异无统计学意义（P>0.05）；HL-1心肌细胞感染组不同时间点细胞活性比较，差异有统计学意义（P<0.05）。MSC未感染组与MSC感染组感染后4、12、24 h细胞活性比较，差异无统计学意义（P>0.05）。MSC未感染组、感染组不同时间点细胞活性比较，差异有统计学意义（P<0.05）。HL-1心肌细胞组病毒滴度大于MSC组（P<0.05）。结论　CVB3能够在HL-1心肌细胞中复制，感染CVB3后HL-1心肌细胞活性降低；但CVB3不能在MSC中复制，且感染CVB3后MSC细胞活性并未明显改变。

关键词: 柯萨奇病毒感染, 肠道病毒B型, 人, 肌细胞, 心脏, 间质干细胞

PENG Jun,WEI Wenjuan,QIAN Zhengming, et al. Impact of Coxsackievirus B3 Infection on HL-1 Cardiomyocytes and Mesenchymal Stem Cells　[J]. Chinese General Practice, 2019, 22(3): 301-305. DOI: 10.12114/j.issn.1007-9572.2018.00.249.
彭俊,魏文娟,钱正明等. 柯萨奇B3病毒感染对HL-1心肌细胞及间充质干细胞的影响研究[J]. 中国全科医学, 2019, 22(3): 301-305. DOI: 10.12114/j.issn.1007-9572.2018.00.249.

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