Notch1表达下调抑制病理性瘢痕成纤维细胞增殖并诱导其凋亡

目的探讨慢病毒介导的Notch1表达下调对病理瘢痕成纤维细胞(PSF)中caspase-9活化水平及线粒体膜电位的影响。方法分离培养增生性PSFs,感染Notch1 siRNA慢病毒和阴性对照慢病毒。分别采用RT-qPCR和Western blot检测细胞中Notch1mRNA和蛋白表达;MTT法检测细胞增殖;PI单染法检测细胞周期;Annexin V-FITC/PI法检测细胞凋亡;Western blot检测细胞周期蛋白D1(cyclin D1)、细胞周期依赖性蛋白激酶4(CDK4)、活化的caspase-3(c-caspase-3)和caspase-9(c-caspase-9)蛋白及胞质和线粒体中细胞色素C(cytochrome C)蛋白;JC-1法检测细胞线粒体膜电位。结果Notch1 siRNA慢病毒感染后的PSFs中Notch1表达水平明显低于阴性对照慢病毒和未感染的PSFs的表达水平(P<0.05)。下调Notch1后的PSFs增殖能力降低(P<0.05);细胞G0/G1期比例升高(P<0.05);细胞凋亡率升高(P<0.05);c-caspase-3和c-caspase-9蛋白表达升高(P<0.05);细胞线粒体膜电位明显下降(P<0.05);细胞中cyclin D1和CDK4蛋白水平降低(P<0.05);胞质内的cytochrome C蛋白含量升高(P<0.05)及线粒体中cytochrome C蛋白水平降低(P<0.05)。结论Notch1表达下调抑制PSFs增殖并诱导凋亡。

Down-regulation of Notch1 inhibits proliferation and induces apoptosis of fibroblasts from pathological scars

Objective To investigate the effect of down-regulation of Notch1 expression mediated by lentivirus on the activation of caspase-9 and mitochondrial membrane potential of fibroblast from pathological scars(PSF).Methods PSFs were isolated and cultured and then infected by Notch1 siRNA lentivirus and negative control lentivirus respectively.The level of Notch1 mRNA and protein was detected by real-time PCR and Western blot,respectively.MTT assay was used to detect cell proliferation;PI monochrome staining was used to detect cell cycle changes.Annexin V-FITC/PI assay was used to detect apoptotic changes;Western blot was used to detect the level of cyclin D1,CDK4,c-caspase-3,c-caspase-9 and cytochrome C protein levels in cytoplasm and mitochondria;JC-1 method was used to detect the mitochondrial membrane potential.Results The expression of Notch1 in PSFs transfected with Notch1 siRNA lentivirus was significantly lower than that in the negative control lentiviruses and the non-transfected PSFs(P<0.05).The proliferation of PSFs decreased after Notch1 was down-regulated(P<0.05);the percen-tage of G0/G1 phase increased(P<0.05);the apoptotic rate increased(P<0.05);the expression of c-caspase-3 and c-caspase-9 protein increased(P<0.05);the mitochondrial membrane potential of the cells decreased significantly(P<0.05);the level of cyclin D1 and CDK4 protein in cells decreased(P<0.05);cytochrome C protein increased in cytoplasm and decreased in mitochondria(P<0.05).Conclusions Down-regulation of Notch1 inhibits proliferation and induces apoptosis of PSF.