纳米二氧化硅诱导巨噬细胞焦亡对肺成纤维细胞的影响

Effect of macrophage pyroptosis induced by nano silica on pulmonary fibroblasts

目的探讨纳米二氧化硅(SiNPs)诱导巨噬细胞焦亡对原代肺成纤维细胞基质金属蛋白酶及成纤维细胞向肌成纤维细胞转分化的影响。方法构建SiNPs致巨噬细胞焦亡模型后,采用纳米高光谱定位巨噬细胞中SiNPs、光学显微镜观察巨噬细胞SiNPs暴露后形态、Western blot检测焦亡相关蛋白表达。通过胰酶和Ⅳ型胶原酶消化法提取小鼠原代肺成纤维细胞;以光学显微镜和免疫荧光技术表征并鉴定原代肺成纤维细胞;体外构建SiNPs诱导巨噬细胞焦亡与原代肺成纤维细胞共培养模型。以正常巨噬细胞上清干预原代肺成纤维细胞作为MF组,焦亡巨噬细胞上清干预原代肺成纤维细胞作为PF组,采用qRT-PCR、Western blot技术分别检测原代肺成纤维细胞中MMP2、MMP9的mRNA水平和蛋白表达,免疫荧光、免疫细胞化学染色检测肌成纤维细胞标志物α-SMA蛋白表达。结果纳米高光谱、形态学观察和Western blot检测结果显示,SiNPs成功诱导J774A.1巨噬细胞发生焦亡,光学显微镜观察发现,与胰酶消化法相比,Ⅳ型胶原酶法提取小鼠原代肺成纤维细胞的纯度更高;免疫荧光结果提示Ⅳ型胶原酶法获得的成纤维细胞波形蛋白呈现典型的微丝结构,细胞骨架结构完整。qRT-PCR结果显示,PF组MMP2和MMP9 mRNA水平较MF组增加(P<0.05),Western blot结果显示,PF组MMP2和MMP9蛋白表达水平较MF组升高(P<0.05),免疫荧光结果显示,与MF组相比,PF组α-SMA红色荧光强度增加,免疫细胞化学染色为强阳性。结论SiNPs诱导巨噬细胞焦亡能够促进原代肺成纤维细胞MMP2和MMP9表达水平上调,并促进成纤维细胞向肌成纤维细胞转分化。

Objective To investigate the effect of silica nanoparticles(SiNPs)induced macrophage pyroptosis on matrix metalloproteinase and fibroblast transdifferentiation to myofibroblasts in primary lung fibroblasts.Methods After constructing the macrophage pyroptosis model induced by SiNPs,the morphological location of SiNPs in macrophages was performed by nano hyperspectral technology,then using optical microscopy to observe macrophage morphology after SiNPs exposure,using Western blot to detect the expression of pyroptosis related protein.Primary lung fibroblasts of mice were extracted by lung-digesting method with trypsin and type Ⅳ collagenase,the characterization and identification of primary lung fibroblasts were performed with optical microscopy and immunofluorescence techniques,respectively.Subsequently,an in vitro co-culture model was constructed to investigate SiNPs-induced macrophage pyroptosis and its interaction with primary lung fibroblasts.The primary lung fibroblasts treated with macrophage culture supernatant was as MF group,while those fibroblast treated with pyroptosis macrophage culture supernatant as PF group,then,qRT-PCR and Western blot techniques were used to detect the expressions of MMP2 and MMP9 at mRNA and protein levels in primary lung fibroblasts,and the expression of myofibroblasts marker α-SMA protein was also detected by immunofluorescence and immunocytochemical staining methods.Results The results of nanometer of hyperspectral imaging,morphological observation and Western blot detection showed that SiNPs successfully induced pyroptosis of J774A.1 macrophages,the optical microscopy observation revealed that compared with trypsin digestion,the purity of primary mice lung fibroblasts extracted by typeⅣcollagenase method was higher,and the immunofluorescence detection results suggested that the Vimentin of fibroblast obtained by type Ⅳ collagenase method exhibited a typical microfilamentous structure,the cytoskeletal structure was complete as well.The qRT-PCR results showed that the mRNA levels of MMP2 and MMP9 were increased in the PF group compared with that of MF group(P<0.05),the Western blot results showed that the protein expression levels of MMP2 and MMP9 were also increased in PF group(P<0.05),the immunofluorescence results showed that the intensity ofα-SMA red fluorescence was increased in the PF group compared with the MF group,and the immunocytochemical staining results showed strongly positive in the PF group.Conclusion The results suggested that SiNPsinduced macrophage pyroptosis may promote the up-regulation of MMP2 and MMP9 expression levels in primary lung fibroblasts as well as the fibroblast transdifferentiation to myofibroblasts.