摘要
目的 研究BEP 2D细胞经辐射诱发恶性转化过程中 ,作为SMAD蛋白家族的抑制分子 ,Smad 7对TGF β SMAD介导信号通路的调控。 方法 用Northernblot检测TGF β刺激之后 ,永生化BEP 2D细胞及辐射诱发恶性转化的BERP 35T 2细胞中Smad 7mRNA的表达水平。人工合成SMAD结合元件 (SBE)重复序列 ,同报告基因碱性磷酸酶融合。构建好的载体同Smad 7真核表达载体共转染 ,TGF β刺激 ,通过报告基因的表达丰度来检测Smad 7对TGF β SMADs介导的信号通路的调控。结果 Northern杂交结果表明 ,永生化细胞Smad 7基因对TGF β刺激的应答正常 ,恶性化细胞的应答降低。基因转染的结果表明 ,永生化细胞中SBE的基础活性较恶性化细胞高 ;TGF β刺激之后 ,永生化细胞中SBE活性显著增强 ,恶性化细胞变化不明显 ;同Smad 7真核表达载体共转染之后 ,两种细胞中SBE活性均显著降低。结论 在辐射诱发细胞发生恶性转化过程中 ,Smad 7基因对TGF β信号通路的反馈调节作用发生紊乱 ,使TGF β信号通路持续处于抑制状态 ,TGF β对细胞的负性调控作用减弱 ,细胞进一步向恶性化发展。
Objective To analyze the regulating effect of Smad 7 gene on TGF-β/SMADs-mediated signal transduction pathway in the process of malignant transformation of immortalized human bronchial epithelial cells BEP2D. Methods Northern blot was used to examine the responsiveness of Smad 7 gene to TGF-β 1 in BEP2D cell line and its malignantly transformed BERP35T2 cell line cells.SBE4s containing four tandem repeats of an 8-bp palindromic consensus Smad-binding elements were ligated to multiple clone sites of pTAL-SEAP,a reporter vector which fused with alkaline phosphatase gene.Transient transfection was performed to investigate the regulation of Smad 7 on TGF-β/SMAD-mediated signal pathway. Results Northern blot results showed that when treated with TGF-β1,expression of Smad 7 gene was up-regulated rapidly in BEP2D cells,reaching a major peak at 60 and then declining at 90 min;but in BERP35T2 cells,it was not obviously increased after that treatment.Transient transfection of SBE4-SEAP reporter vector into BEP2D and BERP35T2 cells showed that the basal activity of the reporter was higher in the former than in the latter.Transient co-transfection of full-length Smad 7 cDNA construct with SBE4-SEAP reporter vector led to decreased activity of the reporter.Furthermore,the responsiveness of SBE to TGF-β1 was more sensitive in BEP2D cells than in BERP35T2 cells. Conclusion In the process of malignant transformation of cells the disorder of Smad7 in negative feedback regulation of TGF-β signaling pathways leads to down-regulation of responsiveness of the cells to TGF-β1,and also weakens the negative modulation activity of TGF-β1 to cells.All these could contribute to further malignant transformation of these cells. ;
出处
《中华放射医学与防护杂志》
CAS
CSCD
北大核心
2003年第5期311-313,共3页
Chinese Journal of Radiological Medicine and Protection
基金
国家重点基础研究发展规划 973项目 (G19980 5 12 0 7)
