Musculin影响小鼠骨髓来源树突状细胞生物学功能的初步研究

Effect of Musculin on biological function of mouse bone marrow-derived dendritic cells

目的探讨新型转录因子Musculin对小鼠骨髓来源树突状细胞(bone marrow-derived dendritic cells,BMDC)生物学功能的影响。方法获取野生型(wildtype,WT)和Musculin敲除(knockout,KO,-/-)小鼠骨髓细胞,用粒细胞-巨噬细胞集落刺激因子(granulocyte-macrophage colony stimulating factor,GM-CSF)和IL-4体外诱导BMDC,7 d后加入脂多糖(lipopolysaccharide,LPS),继续培养24 h;动态观察细胞形态变化,收集WT组和Musculin-/-组BMDC及上清,流式细胞术分析各组细胞表达CD11c、CD40、CD80、CD86、PD-L1和IAIE的比例,CCK-8法检测各组细胞刺激异源淋巴细胞增殖情况,并计算刺激指数(stimulation indexes,SI),ELISA法检测各组细胞上清中IL-1β、IL-6、IL-10、IL-12(P40)、IL-12(P70)和TNF-α的水平。结果与WT组比较,Musculin-/-组BMDC形态相似,纯度无明显差异;表面共刺激分子CD40、CD80和CD86的比例显著下降(P<0.05),共抑制分子PD-L1和MHC-Ⅱ分子IAIE的比例也降低,但差异无统计学意义;刺激异源淋巴细胞后的数量和刺激指数上升,尤其是刺激5 d以后(P<0.05);上清中促炎因子IL-1β、IL-6、IL-12(P40)、IL-12(P70)和TNF-α水平显著升高(P<0.05),但抗炎因子IL-10的水平变化不明显。结论Musculin可能增强BMDC介导的免疫功能并抑制炎症反应,提示Musculin能够调控BMDC的生物学功能。

ObjectiveTo investigate the effect of a novel transcription factor,Musculin,on the biological functions of bone marrow-derived dendritic cells(BMDC)in mice.MethodsBone marrow cells from wildtype(WT)and Musculin knockout(KO,-/-)mice were obtained and cultured with granulocyte-macrophage colony stimulating factor(GM-CSF)and IL-4 so as to induce BMDC in vitro.After 7 d,the BMDC were added with lipopolysaccharide(LPS)and continued to culture for 24 h.Besides the cellular morphological changes were observed dynamically,BMDC and the supernatants were collected from the WT and Musculin-/-cells.Flow cytometry was used to analyze the proportion of positive cells of CD11c,CD40,CD80,CD86,PD-L1 and IAIE in each group.CCK-8 assay was adopted to detect the cellular proliferation for stimulating heterologous lymphocytes and the different stimulation indexes(SI)were calculated respectively.ELISA was applied to detect the supernatant contents of IL-1β,IL-6,IL-10,IL-12(P40),IL-12(P70)and TNF-αin each group.ResultsThe BMDC from the Musculin-/-group had similar morphology and no significant difference in purity,decreased percentages of co-stimulatory molecules such as CD40,CD80 and CD86(P<0.05),and reduced percentages of co-inhibitory molecules PD-L1 and major histocompatibility complex classⅡ(MHC-Ⅱ)IAIE though no significant differences when compared with those of WT group.Moreover,the number of heterologous lymphocytes and related SI after of mixed lymphocyte reaction(MLR)were obvious higher in the Musculin-/-group than the WT group,especially after 5 days’stimulation(P<0.05).In addition,the supernatant levels of proinflammatory factors including IL-1β,IL-6,IL-12(P40),IL-12(P70)and TNF-αwere significantly increased in the Musculin-/-group than the WT group(P<0.05),but no such changes were observed in anti-inflammatory factor IL-10 level between the 2 groups.ConclusionMusculin may enhance the immune function mediated by BMDC and suppress inflammatory reaction,suggesting that it can regulate the biological functions on BMDC.