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目的探讨肿瘤坏死因子-α(TNF-α)激活核因子-κB(NF-κB)信号通路对成纤维细胞生物学行为的影响。方法小鼠胚胎成纤维细胞(MEF)进行体外培养,将细胞分为TNF-α组、TNF-α+吡咯烷二硫氨基甲酸(PDTC)组及对照组,处理后的细胞采用CCK-8方法检测细胞增殖情况、Western blot和细胞免疫荧光检测IκBα蛋白表达与定位、ELISA检测基质细胞衍生因子-1α(SDF-1α)的分泌。结果 TNF-α在一定浓度范围内对成纤维细胞起到明显的促增殖作用,CCK-8检测显示TNF-α组在不同浓度和不同时相点MEF的光密度值与TNF-α+PDTC组及对照组相比差异显著(P<0.05);免疫荧光染色显示IκBα主要分布于细胞质,TNF-α组与对照组和TNF-α+PDTC组相比,IκBα荧光强度明显减弱。Western blot结果发现TNF-α刺激后30 min,TNF-α组和TNF-α+PDTC组IκBα蛋白表达[(3 070.0±39.0)vs(1 421.7±75.3),(3 212.2±57.5)vs(1 468.2±45.8),P<0.05]均降至最低值,随时间推移逐渐恢复,TNF-α+PDTC组IκBα蛋白表达显著高于TNF-α组[(3 112.3±89.7)vs(2 610.4±26.9),P<0.05];ELISA检测发现TNF-α作用后成纤维细胞SDF-1α的分泌呈减少趋势[(15.9±1.6)vs(12.7±0.6),P<0.05],TNF-α+PDTC组则能显著逆转TNF-α作用[(23.5±3.2)vs(15.0±1.2),(21.6±0.4)vs(12.7±0.6),P<0.05],促进SDF-1α分泌。结论 TNF-α能通过激活NF-κB通路,影响小鼠胚胎成纤维细胞的增殖并抑制SDF-1α分泌。
Objective To investigate the effects of tumor necrosis factor-α (TNF-α) activating nuclear factor-κB (NF-κB) signaling pathway on the biological behavior of fibroblasts. Methods Mouse embryonic fibroblasts (MEF) were cultured in vitro. The cells were divided into TNF-α group, TNF-α + pyrrolidine dithiocarbamate group and control group. The treated cells were treated with CCK-8 The cell proliferation was detected by Western blot and immunofluorescence. The expression of IκBα protein was detected by Western blot and the secretion of stromal cell-derived factor-1α (SDF-1α) was detected by ELISA. Results The concentration of TNF-α promoted the proliferation of fibroblasts in a certain concentration range. CCK-8 showed that the optical density value of MEF at different concentrations and different time points in TNF-α group was significantly lower than that in TNF-α + PDTC group (P <0.05). Immunofluorescence staining showed that IκBα mainly distributed in the cytoplasm. The fluorescence intensity of IκBα in TNF-α group was significantly decreased compared with control group and TNF-α + PDTC group. Western blot showed that the expression of IκBα in TNF-α and TNF-α + PDTC groups was significantly higher than that in TNF-α and PDTC groups at 30 min after stimulation of TNF-α [(3 070.0 ± 39.0 vs 1 421.7 ± 75.3 vs (3 212.2 ± 57.5 vs vs 468.2 ± 45.8), P <0.05] all decreased to the lowest values and gradually recovered with time. The expression of IκBα in TNF-α + PDTC group was significantly higher than that in TNF-α group [(3 112.3 ± 89.7) vs (610 610 ± 26.9) ), P <0.05]. ELISA showed that the secretion of SDF-1α in fibroblasts was decreased by TNF-α [(15.9 ± 1.6) vs (12.7 ± 0.6), P <0.05] (23.5 ± 3.2) vs (15.0 ± 1.2), (21.6 ± 0.4) vs (12.7 ± 0.6), respectively, P <0.05], and promoted SDF-1α secretion. Conclusion TNF-α can affect the proliferation of mouse embryonic fibroblasts and inhibit the secretion of SDF-1α by activating NF-κB pathway.