【摘要】 目的 探讨冷刺激对小鼠肺泡巨噬细胞（MH-S细胞）表型的影响。方法 将MH-S细胞置37℃培养24 h，于36、34、32℃分别冷刺激0、0.5、1、3、6、9、12 h,qRT-PCR法检测MH-S细胞中炎性因子白介素-1β(interleukin-1β,IL-1β)及白介素-10(interleukin-10,IL-10)基因mRNA转录水平。将MH-S细胞置37℃培养24 h，于34℃冷刺激0.5 h,qRT-PCR法检测MH-S细胞中肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）、精氨酸酶（Arginase1,Arg1）基因mRNA转录水平（以冷刺激0 h为对照）；免疫荧光法检测iNOS和Arg1的表达情况（以37℃培养0.5 h的MH-S细胞为阴性对照）；Western blot法检测iNOS、TNF-α及核因子-κB(nuclear factor-kappa B,NF-κB)的表达水平（以37℃培养0.5 h的MH-S细胞为阴性对照）。结果 34℃培养0.5 h条件下，MH-S细胞中IL-1β及IL-10基因mRNA转录水平最高，因此采用该条件建立MH-S细胞的冷刺激模型。与0 h比较，34℃培养0.5 h的MH-S细胞中iNOS、TNF-α和Arg1基因mRNA转录水平均明显升高（t分别为3.733、12.190、6.793,P均<0.05）。与阴性对照组比较，刺激组MH-S细胞中iNOS及Arg1的荧光表达强度均有所增强，其中iNOS增强更为明显。与阴性对照组比较，刺激组MH-S细胞中iNOS和TNF-α蛋白表达水平升高，但差异无统计学意义（t分别为0.675和1.514,P均> 0.05）;NF-κB表达水平明显升高（t=3.092,P <0.05）。结论 34℃冷刺激0.5 h可提高MH-S细胞中IL-1β、IL-10、TNF-α、iNOS、Agr1、NF-κB等炎性相关因子的表达，可激活MH-S细胞中的NF-κB信号通路，诱导炎症蛋白表达，促进细胞活化。更多还原

【Abstract】 Objective To investigate the effect of cold stimulation on the phenotype of alveolar macrophages(MH-S cells) in mice. Methods MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 36,34 and 32 ℃ for 0,0. 5,1,3,6,9 and 12 h respectively. The mRNA transcription levels of interleukin-1β(IL-1β) and interleukin-10(IL-10) genes in MH-S cells were detected by qRT-PCR. MH-S cells were cultured at 37 ℃ for 24 h,and cold stimulated at 34 ℃ for 0. 5 h,which were detected for the mRNA transcription levels of tumor necrosis factor-α(TNF-α),inducible nitric oxide synthase(iNOS)and Arginase1(Arg1)genes by qRT-PCR(MH-S cells with 0 h cold stimulation as control),detected for the expression of iNOS and Arg1 by immunofluorescence assay(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control)and detected for the expression levels of iNOS,TNF-α and nuclear factor-kappa B(NF-κB)by Western blot(MH-S cells cultured at 37 ℃ for 0. 5 h as negative control). Results The mRNA transcription levels of IL-1β and IL-10 genes in MH-S cells were the highest when the cells were cultured at 34 ℃ for 0. 5 h,therefore,the cold stimulation model of MH-S cells was established under this condition. Compared with the cells cultured for 0 h,the mRNA transcription levels of iNOS,TNF-α and Arg1genes in MH-S cells cultured at 34 ℃ for 0. 5 h increased significantly(t = 3. 733,12. 190 and 6. 793,respectively,each P < 0. 05). Compared with the negative control group,the fluorescence expression intensity of iNOS and Arg1 in MH-S cells in the stimulation group increased,especially iNOS,the expression levels of iNOS and TNF-α proteins increased with no significant difference(t = 0. 675 and 1. 514,respectively,each P > 0. 05),and the expression level of NF-κB increased significantly(t = 3. 092,P < 0. 05). Conclusion Cold stimulation at 34 ℃ for 0. 5 h can increase the expression of inflammatory factors such as IL-1β,IL-10,TNF-α,iNOS,Agr1 and NF-κB in MH-S cells,activate NF-κB signaling pathway in MH-S cells,induce the expression of inflammatory proteins and promote cell activation.更多还原