小鼠病毒性心肌炎CXCL13表达及其意义

Expression and significance of CXCL13 in mouse viral myocarditis

目的:检测病毒性心肌炎(VMC)小鼠急性期6个时间点CXCL13的表达,了解其与VMC发病的关系。方法:雄性Balb/c小鼠腹腔注射柯萨奇病毒B3(CVB3)建立小鼠VMC模型为VMC组,腹腔注射等量PBS为对照组,采用实时荧光定量聚合酶链反应(RT-qPCR)和免疫组化的方法检测第0周、第1周、第2周、第3周、第4周和第6周心肌组织中CXCL13 mRNA和CXCL13蛋白的表达量。二次磁珠分选出VMC小鼠CD4+CXCR5+T细胞(Tfh细胞),磁珠分离出未注射病毒的同龄正常BALB/c小鼠初始CD19^+B细胞,分别接种于transwell上室及下室,在下室培养液中加或不加CXCL13(100 ng/mL)进行共培养,5 d后ELISA检测上清液中抗腺嘌呤核苷酸转位子(ANT)抗体的浓度。结果:与对照组比较,VMC组小鼠自感染CVB3第1周心肌中CXCL13 m RNA的表达升高,第2周时表达最强,并一直持续到第6周,VMC组各周相对表达量均高于相应的对照组(P<0.05)。VMC组小鼠自1周起,心肌CXCL13呈现阳性表达,第2周时达峰值,并维持高水平至第6周(P<0.05)。CXCL13刺激后,Tfh细胞与B细胞分室共培养的上清液中抗ANT抗体升高(P<0.05)。结论:CXCL13在VMC小鼠心肌组织中高表达,并可增加VMC中抗ANT抗体的浓度,提示CXCL13与VMC存在密切关系,CXCL13有可能成为减轻VMC中自身抗体形成的新的治疗靶点。

Objective:To detect the expression of CXCL13 at 6 time points during the acute phase of viral myocarditis(VMC)in mice,and to explore its relationship with the incidence of VMC.Methods:Male Balb/c mice were intraperitoneally injected with Coxsackie virus B3(CVB3)to establish the mouse VMC model and serve as the VMC group,while the control group were intraperitoneally injected with the same amount of PBS.The expression of CXCL13 mRNA and protein in myocardial tissues at week 0,week 1,week 2,week 3,week 4,and week 6 were detected by using quantitative polymerase chain reaction(RT-qPCR)and immunohistochemistry.The CD4+CXCR5+T cells(Tfh cells)of VMC mice were sorted out by secondary magnetic beads,and the original CD19+B cells of normal BALB/c mice of the same age without virus injection were isolated by magnetic beads.CD4+CXCR5+T cellsand CD19+B cellswereseeded in the upper and lower chambers of Transwell,respectively.In the lower chamber,co-culture was performed with or without CXCL13(100 ng/mL)in the culture medium.After 5 days,the concentration of anti-adenine nucleotide transposon(ANT)antibody in the supernatant was detected by ELISA.Results:Compared with the control group,the expression of CXCL13 mRNA in the myocardium of the VMC group was increased since week 1 after CVB3 infection,the expression was strongest at week 2 and remained at a high level until week 6.In each week,the relative expression of CXCL13 mRNAin the test group was always higher than the control group(P<0.05).The mice in the VMC group had positive expression of CXCL13 in the myocardium since week 1,which reached thepeaked at week 2 and maintained a high level until week 6(P<0.05).After CXCL13 stimulation,the anti-ANT antibody in the supernatant of Tfh cells co-cultured with B cells was increased(P<0.05).Conclusion:CXCL13 is highly expressed in the myocardial tissue of VMC mice and can increase the concentration of anti-ANT antibody in VMC,which suggests that CXCL13 is closely related to VMC.Thus,CXCL13 may become a new therapeutic target to reduce the formation of autoantibodies in VMC.