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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 study the regulation of TGFβ SMAD-mediated signal pathway by Smad 7 as a suppressor of the SMAD protein family during radiation-induced malignant transformation of BEP 2D cells. Methods Northern blot was used to detect the expression of Smad 7 mRNA in immortalized BEP 2D cells and irradiated malignant transformed BERP 35T 2 cells after TGF β stimulation. Synthetic SMAD binding element (SBE) repeats were fused to the reporter alkaline phosphatase. The constructed vector was co-transfected with the Smad7 eukaryotic expression vector and stimulated by TGF-β. The expression of Smad 7 in TGFβ SMADs-mediated signal pathway was detected by reporter gene abundance. Results The result of Northern blot showed that Smad 7 gene of immortalized cells responded to TGF-β normally and the response of malignant cells decreased. The results of gene transfection showed that the basal activity of SBE in immortalized cells was higher than that of malignant cells. The SBE activity in immortalized cells was significantly increased and the malignant cells did not change obviously after stimulation with TGF-β. After transfection, SBE activity was significantly reduced in both cells. Conclusions During the process of radiation-induced malignant transformation, the Smad 7 gene has a disturbed effect on the feedback regulation of TGF-β signaling, which inhibits the TGF β signaling pathway. TGF-β negatively regulates the cells negatively, Malignant development.