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目的探讨激活素(Act)A细胞内高表达对Act A/Smads通路基础活化水平的影响。方法以ActβA重组载体(Act A-p IRES2-EGFP)和空载体(p IRES2-EGFP)稳定转染的PC12细胞为阳性和阴性转染组,正常PC12细胞为对照,利用实时荧光定量聚合酶链反应(RT-PCR)、Western印迹法检测3组细胞内Act A、Smad3和磷酸化Smad3在转录及蛋白水平的表达,激光共聚焦观察Smad4的质核分布情况。结果与阴性转染组相比,阳性转染组Act A mRNA及蛋白表达分别提高4.5和1倍,Smad3总蛋白及磷酸化蛋白表达分别提高了36.4%和86.9%,Smad4蛋白在阳性转染组细胞核内分布增加。结论 Act A内源性高表达可提高PC12细胞内Act A/Smads通路的基础活化水平。
Objective To investigate the effect of Act A high intracellular expression on basal activation of Act A / Smads pathway. Methods PC12 cells stably transfected with ActβA-EGFP and pIRES2-EGFP were positive and negative, and normal PC12 cells were control. RT-PCR). Western blotting was used to detect the expression of Act A, Smad3 and phosphorylated Smad3 in the three groups of cells. The distribution of Smad4 nucleus was observed by confocal laser scanning microscope. Results Compared with the negative transfection group, the positive expression of Act A mRNA and protein were increased by 4.5 and 1 fold, respectively, and the expression of Smad3 total protein and phosphorylated protein were increased by 36.4% and 86.9% respectively. The positive expression of Smad4 protein in positive transfection group Increased distribution within the nucleus. Conclusions Endogenous high expression of act A enhances basal activation of Act A / Smads pathway in PC12 cells.