TSG-6促进瘢痕疙瘩成纤维细胞凋亡及其NF-κB信号通路影响的实验研究

Study on the effects of TSG-6 on the apoptosis of keloid fibroblasts and NF-κB signaling

目的探讨TSG-6预处理对瘢痕疙瘩成纤维细胞凋亡及NF-κB信号通路的影响。方法标本人瘢痕疙瘩共3例,采用酶消化法分离纯化得到人瘢痕疙瘩成纤维细胞(KFs),MTT法检测rh TSG-6蛋白对KFs的增殖抑制作用,计算半数抑制浓度(IC50),流式细胞术检测KFs(空白对照组)与接近IC50rh TSG-6蛋白处理组(rh TSG-6实验组)细胞凋亡情况。Western blot检测各组细胞中IκBα、p-IκBα、活化caspase3及caspase8的表达。凝胶电泳迁移率实验(EMSA)检测各组细胞NF-κB DNA结合活性。结果 rh TSG-6在体外对KFs增殖有明显抑制作用(IC50接近300 ng/ml),细胞凋亡率增高,差异有统计学意义(P<0.05);Western blot法检测显示rh TSG-6实验组KFs中IκBα表达增加,p-IκBα表达减少,活化caspase3和caspase8表达明显增多。EMSA结果显示rh TSG-6实验组NF-κB DNA结合活性明显降低。结论TSG-6在体外可能通过抑制NF-κB信号通路活性进而抑制KFs增殖并促进其凋亡。

Objective To investigate the effects of tumor necrosis keloid fibroblasts and detect NF-κB signaling pathway. Methods factor-stimulated gene 6 （TSG-6） on apoptosis of Fibroblasts derived from keloid were cultured with different concentration of rhTSG-6 （0,50,100,200,400 ng/ml） in vitro. MTT assay was used to detect the in- hibitory effect of rhTSG-6 on the proliferation of KFs, and then IC50 value was calculated. Cell apoptosis was deter-mined by flow cytometry. The expression levels of IκBα, p-IκBα, cleaved-Caspase3 and Caspase8 were determined by Western blot, respectively. NF-κB activity was assessed by eleetrophonetic mobility shift assay （EMSA）. Re- suits rhTSG-6 showed proliferation inhibition effect against KFs and ICs0 value of rhTSG-6 to KFs was nearly 300 ng/ml. After treated with rhTSG-6 at the concentration of 300 ng/ml for 48 h, the rate of apoptosis of KFs was in- creased （P 〈 0.05）. The expression level of cleaved-caspase 3 and easpase 8 were significantly increased in rhTSG-6 treated group. In the other hand, the whole p-IκBα level was significantly reduced upon rhTSG-6 treat- ment with marked increase in IκBα levels. NF-κB activity was decreased significantly compared to the control group. Conclusion The data suggest that TSG-6 may induce cell apoptosis in KFs via inhibiting the activation of NF-κB pathway.