哮喘小鼠中髓样抑制细胞及Arg-1、iNOs的表达

Expression of MDSCs,Arg-1 and iNOs in asthmatic mice

目的:探讨哮喘小鼠脾细胞中髓样抑制细胞(MDSCs)两个亚群所占有核细胞比例,以及在小鼠中精氨酸酶-1(Arg-1)和诱导性一氧化氮合酶(iNOs)的表达水平。方法:将6~8周清洁级BALB/c雄性小鼠随机分为对照组(control)和哮喘组(OVA),给予哮喘组OVA致敏和激发,建立哮喘小鼠模型,用PBS代替OVA给予对照组。最后1次激发后24 h内检测支气管肺泡灌洗液(BALF)中细胞总数及细胞分类计数;采用酶联免疫吸附法(ELISA)测定小鼠血清中抗卵清蛋白(OVA)特异性IgE水平;小鼠肺组织HE染色,光镜下观察病理变化;流式细胞术检测小鼠脾细胞中MDSCs两个亚群所占有核细胞比例;用荧光定量PCR(qRT-PCR)技术检测小鼠脾细胞及肺组织中MDSCs产物Arg-1、iNOs mRNA的表达水平;用Western blot方法检测小鼠肺组织中MDSCs产物Arg-1、iNOs蛋白表达。结果:哮喘组小鼠肺部可见明显的炎性细胞浸润,与对照组相比,哮喘组BALF中细胞总数、嗜酸性粒细胞百分比及血清抗OVA特异性IgE明显增高(P<0.05);哮喘组小鼠脾细胞中G-MDSC亚群细胞所占比例明显升高(P<0.001),而M-MDSC亚群无明显变化(P>0.05);哮喘组小鼠脾细胞及肺组织中Arg-1 mRNA表达均增加(P<0.05),而iNOs mRNA表达均减少(P<0.05);哮喘组小鼠肺组织中Arg-1 蛋白表达增加(P<0.05),而iNOs蛋白表达减少(P<0.05)。结论:哮喘组小鼠中的MDSCs,尤其是G-MDSC可能通过产生高水平的Arg-1参与哮喘的发病。

Objective:To detected the expression of myeloid-derived suppressor cells(MDSCs),Arg-1 and iNOs in asthmatic mice.Methods:BALB/c mice were randomly divided into control and OVA groups,mice in the OVA group were sensitized and challenged with OVA,while mice in the control group with PBS replaced OVA.Within 24 hours after the final OVA challenge,we tested the number of total cells and the eosinophils(EOS) counts in bronchoalveolar lavage fluid(BALF) by light microscopy.The accumulation of Anti-OVA-specific IgE in serum of mice was measured by ELISA.The severity of inflammation in lung tissue was evaluated by hematoxylin and eosin (HE) staining.The percentage of MDSCs were analyzed by flow cytometry.The expression levels of Arg-1 and iNOs mRNA were determined by quantitative Real time polymerase chain reaction(qRT-PCR).The protein expression levels of Arg-1 and iNOs were detected by Western blot.Results:We found that the inflammation of lung tissue in OVA group was more significant than that in control group.The number of total cells and the proportion of eosinophils in BALF of OVA group were significantly higher than that of control group.The accumulation of Anti-OVA-specific IgE in serum of OVA group was higher than that in control group(P<0.05).The spleen cells of OVA group showed a higher percentage of G-MDSC compared with control group(P<0.001),but,for the percentage of M-MDSC,there was no significant change in the two groups(P>0.05).Both in lung tissue and in the spleen cells,the expression levels of Arg-1 mRNA from OVA group was increased significantly compared with control group(P<0.05),and iNOs mRNA expression was obviously lower than in the control group(P<0.05).Expression of Arg-1 protein in lung tissue was increased(P<0.05),and expression of iNOs protein was decreased(P<0.05).Conclusion:MDSCs in asthmatic mice,especially G-MDSC,may regulate to the progression of the asthma through producing high levels of Arg-1.