沉默Fcgr3降低SiO_(2)诱导的小鼠肺泡巨噬细胞系MH-S炎性因子表达

Silencing Fcgr3 decreases SiO_(2)-induced inflammatory cytokines expression in mouse alveolar macrophage cell line MH-S

目的探讨沉默免疫球蛋白G Fc段受体Ⅲ(FcγRⅢ)对SiO_(2)诱导的小鼠肺泡巨噬细胞系MH-S炎性因子表达的影响。方法构建Fcgr3 shRNA的慢病毒质粒;人胚肾细胞系293T培养包装产生慢病毒;慢病毒感染沉默MH-S中的FcγRⅢ,并筛选稳定感染的细胞。定量多聚酶链反应(qPCR)和蛋白免疫印迹法(Western blot)检测MH-S巨噬细胞系中FcγRⅢ的mRNA和蛋白表达。将MH-S细胞分为MH-S+sh-NC、MH-S+SiO_(2)+sh-NC、MH-S+sh-Fcgr3和MH-S+SiO_(2)+sh-Fcgr3,其中SiO_(2)处理条件为100 mg/L连续刺激12 h。通过qPCR检测炎性因子转录水平,酶联免疫吸附测定(ELISA)检测细胞培养上清中的炎性因子的含量,包括肿瘤坏死因子(TNF-α)、白介素1β(IL-1β)和白介素6(IL-6)。结果包装慢病毒并感染细胞后建立稳定沉默Fcgr3的MH-S细胞系,稳定转染细胞中FcγRⅢ的转录与蛋白表达水平较对照组降低(P<0.05)。与对照组相比,SiO_(2)刺激后Tnf、Il1b和Il6的转录水平明显升高(P<0.05),沉默Fcgr3后炎症因子转录水平显著下降(P<0.05);与对照组相比,SiO_(2)刺激后TNF-α和IL-6的分泌量明显升高(P<0.05),沉默Fcgr3后炎症因子分泌水平显著下降(P<0.05)。结论沉默Fcgr3能够降低SiO_(2)诱导的肺泡巨噬细胞炎性因子的表达。

Objective To investigate the effect of receptorⅢfor the Fc region of immunoglobulin G(FcγRⅢ)on SiO_(2)-induced inflammatory factors expression in the mouse alveolar macrophage cell line MH-S.Methods The lentivirus plasmids with Fcgr3 shRNA were constructed;human embryonic kidney cell line 293T was incubated for packaging of the lentivirus for silencing FcγRⅢin MH-S.Quantitative polymerase chain reaction(qPCR)and Western blot were used to detect the transcription level and protein expression of FcγRⅢin MH-S.MH-S cells were divided into MH-S+sh-NC,MH-S+SiO_(2)+sh-NC,MH-S+sh-Fcgr3,MH-S+SiO_(2)+sh-Fcgr3,and the SiO_(2) stimulation condition was incubation in a concentration of 100 mg/L for 12 h.Transcription levels of inflammatory cytokines was detected by qPCR,and the content of inflammatory cytokines in cell culture supernatants were detected by enzyme-linked immunosorbent assay(ELISA),including tumor necrosis factor(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6).Results The stable Fcgr3-silencing cell line was established after lentivirus infection.Transcription level and protein expression of FcγRⅢin stable Fcgr3-silencing cell line were lower than that in control group(P<0.05).The transcription level of Tnf,Il1b and Il6 was significantly increased after SiO_(2) stimulation as compared with the control group(P<0.05),and significantly decreased after Fcgr3 silencing(P<0.05).The secretion of TNF-αand IL-6 was obviously increased after SiO_(2) stimulation(P<0.05),and significantly decreased after Fcgr3 silencing(P<0.05).Conclusions Silencing Fcgr3 may decrease the expression of inflammatory cytokines induced by SiO_(2) in alveolar macrophages.