游离SiO_(2)粉尘对小鼠BMDCs细胞因子表达影响的体外实验

Effects of free silica dust on cytokine expression of mouse myeloid dendritic cells in vitro

目的观察游离二氧化硅(silicon dioxide,SiO_(2))粉尘对树突状细胞(dendritic cells,DCs)细胞因子表达的作用,在适应性免疫应答初始环节探讨游离SiO_(2)粉尘致肺部炎症和矽肺发生的可能作用机制。方法体外诱导培养小鼠骨髓源树突状细胞(bone marrow-derived dendritic cells,BMDCs),分别设为对照组、脂多糖(lipopolysaccharide,LPS,25ng/mL)阳性活化组、SiO_(2)(10μg/cm^(2))+LPS处理组、SiO_(2)(25μg/cm^(2))+LPS处理组、SiO_(2)(50μg/cm^(2))+LPS处理组、SiO_(2)(75μg/cm^(2))+LPS处理组、SiO_(2)(150μg/cm^(2))+LPS处理组。具体处理方式为不同浓度SiO_(2)预处理BMDCs 2h后,加入25 ng/mL LPS继续孵育6h或24h,利用Cell Counting Kit-8(Cell Counting Kit-8,CCK8)试剂盒测定BMDCs细胞活力,酶联免疫吸附法(enzyme-linked immuno sorbent assay,ELISA)测定BMDCs细胞上清液中细胞因子含量,实时荧光定量PCR(real-time fluorescence quantitative PCR,Real-time qPCR)法检测BMDCs基因表达水平。结果与对照组相比,LPS阳性活化组增强BMDCs的细胞活力,差异有统计学意义(P<0.05)。与LPS阳性活化组相比,75和150μg/cm^(2)抑制BMDCs的细胞活力,且差异均有统计学意义(P<0.05),其余各浓度SiO_(2)未对BMDCs细胞活力产生明显影响。与对照组相比,LPS阳性活化组BMDCs中肿瘤坏死因子(tumor necrosis factor,TNF)-α、白介素(interleukin,IL)-6和IL-1β的蛋白和基因表达水平显著升高,且差异均有统计学意义(P<0.05)。与LPS阳性活化组相比,10、25和50μg/cm^(2) SiO_(2)处理组BMDsC中TNF-α蛋白表达和IL-6基因表达水平显著升高;25和50μg/cm^(2) SiO_(2)促进LPS活化的BMDCs分泌炎症因子IL-6,而10μg/cm^(2) SiO_(2)则抑制其分泌IL-6,且差异均具有统计学意义(P<0.05)。与对照组相比,LPS阳性活化组BMDCs中IL-12、IL-10和IL-23的蛋白和基因表达水平显著升高,且差异均有统计学意义(P<0.05)。与LPS阳性活化组相比,10和50μg/cm^(2) SiO_(2)抑制LPS活化的BMDCs分泌IL-12,10μg/cm^(2) SiO_(2)抑制其分泌IL-10,而25μg/cm^(2) SiO_(2)促进其分泌IL-10,且差异均有统计学意义(P<0.05)。与LPS阳性活化组相比,10和25μg/cm^(2) SiO_(2)抑制LPS活化的BMDCs中炎症因子IL-12b和IL-23a基因表达,25和50μg/cm^(2) SiO_(2)抑制其IL-10基因表达,且差异均具有统计学意义(P<0.05)。与LPS阳性活化组相比,10和25μg/cm^(2) SiO_(2)促进LPS活化的BMDCs分泌IL-23,25μg/cm^(2) SiO_(2)促进其分泌细胞因子转化生长因子(transforming growth factor,TGF)-β,且差异均有统计学意义(P<0.05)。结论游离SiO_(2)粉尘可促进小鼠BMDCs分泌炎症因子TNF-α、IL-23和TGF-β,同时抑制BMDCs分泌子IL-12。

Objective To observe effects of free silica(SiO_(2))dust on the expression of cytokines in dendritic cells(DCs),the strongest ability of antigen presentation,and to explore the possible mechanism of pulmonary inflammation andsilicosis induced by free SiO_(2) dust from the point of view of adaptive immune response.Methods Mouse bone marrow-derived dendritic cells(BMDCs)were induced and cultured in vitro,which were divided into control group,lipopolysaccharide(LPS)positive activation group(25 ng/mL),SiO_(2)(10μg/cm^(2))+LPS treatment group,SiO_(2)(25μg/cm^(2))+LPS treatment group,SiO_(2)(50μg/cm^(2))+LPS treatment group,SiO_(2)(75μg/cm^(2))+LPS treatment group,SiO_(2)(150μg/cm^(2))+LPS treatment group.The specific treatment method was that BMDCs were pretreated with different concentrations of SiO_(2) for 2 h,then 25 ng/mL LPS was added to incubate for 6 h or 24 h.The viability of BMDCs was determined by Cell Counting Kit-8(CCK8)kit,the cytokines in the supernatant of BMDCs were determined by enzyme linked immunosorbent assay(ELISA),and the gene levels of BMDCs were detected by real-time fluorescence quantitative PCR(Real-time qPCR).Results Compared with the control group,25 ng/mL LPS enhanced the cell viability of BMDCs,and the difference was statistically significant.Compared with the LPS-activated BMDCs,75 and 150μg/cm^(2) SiO_(2) inhibited the cell viability of BMDCs,and the difference was statistically significant(P<0.05),other concentrations of SiO_(2) did not have effects on cell viability of BMDCs.Compared with the control group,the protein and gene levels of tumor necrosis factor(TNF)-α,interleukin(IL)-6 and IL-1βin LPS-activated BMDCs were significantly higher than those of the control group.Compared with LPS-activated BMDCs,the expression of TNF-αprotein and Il-6 gene in BMDCs increased significantly in BMDCs treated with 10,25 and 50μg/cm^(2) SiO_(2).25 and 50μg/cm^(2) SiO_(2) promoted the secretion of inflammatory factor IL-6,10μg/cm^(2) SiO_(2) inhibited the secretion of inflammatory factor IL-6 in LPS-activated BMDCs,and the difference was statistically significant(P<0.05).Compared with the control group,the protein and gene levels of helper T lymphocytes(Th)1-inducible cytokine IL-12,Th2-inducible cytokine IL-10 and Th17-inducible cytokine IL-23 in LPS-activated BMDCs were significantly increased,and the differences were statistically significant.Compared with LPS-activated BMDCs,10 and 50μg/cm^(2) SiO_(2) inhibited secretion of IL-12 in LPS-activated BMDCs,10μg/cm^(2) SiO_(2) inhibited secretion of IL-10 in LPS-activated BMDCs,25μg/cm^(2) SiO_(2) enhanced secretion of IL-10 in LPS-activated BMDCs,and the difference was statistically significant(P<0.05).Compared with LPS-activated BMDCs,10 and 25μg/cm^(2) SiO_(2) inhibited the gene expression of inflammatory cytokine IL-12b and IL-23a in LPS-activated BMDCs,as well as 25 and 50μg/cm^(2) SiO_(2) inhibited the gene expression of inflammatory cytokine IL-10 in LPS-activated BMDCs.Compared with LPS-activated BMDCs,10μg/cm^(2) SiO_(2) and 25μg/cm^(2) SiO_(2) enhanced secretion of Th17 inducible cytokine IL-23 in LPS-activated BMDCs,25μg/cm^(2) SiO_(2) enhanced secretion of regulatory T lymphocyte(Treg)inducible cytokine transforming growth factor(TGF)-βin LPS-activated BMDCs,and the difference was statistically significant(P<0.05).Conclusion Free SiO_(2) dust could promote the secretion of inflammatory cytokines TNF-α,Th17-inducible cytokine IL-23 and Treg-inducible cytokine TGF-β,as well as inhibit the secretion of Th1-inducible cytokine IL-12 in murine BMDCs.