p53/p21信号转导通路在石英诱导人胚肺成纤维细胞恶性转化中的作用

目的观察石英诱导恶性转化的人胚肺成纤维细胞(T-HELF)中p53蛋白的表达量,并探讨p53蛋白对p21、细胞周期蛋白D1和细胞周期蛋白依赖激酶4(CDK4)蛋白表达量及蛋白间相互作用的影响。方法利用胞浆胞核分离技术,分别提取人胚肺成纤维细胞(HELF)、T-HELF胞浆和胞核蛋白,运用Western blot检测p53、磷酸化p53及p21蛋白在细胞中的表达量及分布。利用RNA干扰技术,在T-HELF中转染2μg p53 siRNA,同时设立转染CMV-neo空白载体质粒的对照组,观察p53、磷酸化p53、p21、细胞周期蛋白D1和CDK4的蛋白表达量及改变。用免疫沉淀方法检测HELF和T-HELF中p21、细胞周期蛋白D1及CDK4蛋白结合物水平的变化,并在T-HELF中分别加入20μmol/L p53化学抑制剂pifithrin-α(PFT-α)和2μg p53转染质粒,观察其对p21、细胞周期蛋白D1及CDK4蛋白间相互作用的影响。结果石英刺激HELF可引起胞核中p53及磷酸化p53蛋白表达量显著增高(P<0.05),T-HELF胞核中p53的蛋白表达量较HELF显著降低(P<0.05)。转染p53 siRNA后,p53蛋白与对照组相比表达下降,p21及细胞周期蛋白D1蛋白表达量增加,差异均有统计学意义(P<0.05),尚未观察到CDK4蛋白表达量的变化(P>0.05)。免疫沉淀结果显示抑制p53的表达可下调p21与细胞周期蛋白D1蛋白复合物的表达量(P<0.05),但尚未观察到其对p21、细胞周期蛋白D1与CDK4蛋白结合的影响(P>0.05)。结论 p53可通过调控p21、细胞周期蛋白D1的蛋白表达和蛋白间相互作用参与石英诱导的恶性转化过程。

Role of p53/p21 signal transduction pathway in the malignant transformation of human embryonic lung fibroblasts induced by quartz

OBJECTIVE To observe the expression of p53 protein and investigate the roles of p53 in the expressions and interactions of p21,cyclin D1 and cyclin dependent kinase 4(CDK4)proteins in malignant transformation of human embryonic lung fibroblasts(T-HELF)induced by quartz.METHODS The cytosolic protein and nuclear protein of both HELF and T-HELF cells were extracted by the separation technique of cytoplasm and nuclei.The distribution and expression of p53,phosphorylated p53 and p21 proteins were detected by Western blot.Based on the RNA interference technique,p53 siRNA was transfected into T-HELF cells to observe the protein expression and change of p53,phosphorylated p53,p21,cyclin D1 and CDK4,while the control group was conducted by transfecting the CMV-neo blank vector into the plasmid.The expression levels of p21,cyclin D1 and CDK4 protein complex in HELF and T-HELF cells were detected by immunoprecipitation.After adding 20μmol/L of p53 chemical inhibitor pifithrin-α(PFT-α)and 2μg of p53 siRNA into T-HELF cells respectively,the effect of p53 protein inhibition on p21,cyclin D1 and CDK4 protein complex was also observed.RESULTS Quartz stimulation of HELF caused a significant increase in the expression of p53 and phosphorylated p53 protein in the nucleus(P<0.05).The protein expression of p53 in the nucleus of T-HELF was significantly lower than that of HELF(P<0.05).After transfection of p53 siRNA,the expression of p53 protein was decreased and the expression of p21 and cyclin D1 protein was increased compared with the control group(P<0.05),while the change of expression in CDK4 was not observed(P>0.05).Additionally,the result of immunoprecipitation showed that the inhibition of p53 expression could down-regulate the expression level of the binding complex between p21 and cyclin D1 protein(P<0.05).However,this effect on p21-CDK4 and cyclin DI-CDK4 protein complex was not observed(P>0.05).CONCLUSION By regulating the expression and protein-protein interaction between p21 and cyclin D1,p53 would participate in quartz-induced malignant transformation.