摘要
目的探索转化生长因子β1（TGF-β1）体外诱导。肾脏成纤维细胞活化过程中自噬的变化情况。方法（1）10μg／LTGF-β1刺激大鼠肾脏成纤维细胞（NRK-49F）不同时间（0、12、24h），倒置显微镜观察细胞形态变化，Western印迹检测细胞活化标志物α-平滑肌肌动蛋白（α-SMA）和I型胶原（Coll）表达情况。（2）不同浓度TGF—β1（0、2、5、10、15、20μg/L）刺激细胞1h，10μg／LTGF-β1刺激细胞不同时间（0min、7min、15min、30min、1h、2h、4h、8h、12h），Western印迹检测自噬标志蛋白微管相关蛋白1轻链3（LC3）、p62及自噬相关蛋白总哺乳动物雷帕霉素靶蛋白（t-mTor）、磷酸化哺乳动物雷帕霉素靶蛋白（p-roTor）、Beclin1表达情况。（3）血清饥饿处理细胞2．5h后，加入10μg/TGF—β1刺激细胞不同时间（0、1、4h），倒置显微镜观察细胞形态变化，Western印迹检测LC3、p62表达水平。结果（1）TGF-β1刺激NRK-49F细胞后，细胞形态逐渐由星状转变为长梭形；随着刺激时间的延长，细胞活化标志物仪．SMA、Coll表达水平也逐渐增加（均P〈0．05）。（2）TGF．B1增加NRK-49F细胞中p62蛋白表达（4h左右达峰值），同时测得细胞中p-roTor-过性增加（约30min时达峰值）、Beclinl表达量逐渐降低（均P〈0．05）。（3）TGF-β1降低了血清饥饿处理后的NRK-49F细胞中LC5-Ⅱ的表达，同时促使细胞p62蛋白表达升高（均P〈0．05）。结论在TGF-β1体外诱导肾脏成纤维细胞活化过程中，细胞自噬活性受到抑制，进而可能促进肾间质纤维化的发生和发展。
Objective To explore the changes of autophagy in the transforming growth factor （TGF）- β1- induced activation of renal fibroblasts in vitro. Methods （1） NRK- 49F cells were cultured with 10 μg/L TGF-131 for different times （0, 12, 24 h） in vitro. Morphological changes of the cells were observed under inverted microscope. The protein expressions of α- smooth muscle actin （α-SMA） and type I collagen （Col I ） in NRK-49F cells were measured by Western blotting. （2） NRK- 49F cells were cultured with 0, 2, 5, 10, 15, 20 μg/L TGF-131 for 1 hour and with 10 μg/L TGF-131 for different times （0 min, 7 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h, 12 h） in vitro. The protein expressions of microtubule- associated protein 1 light chain 3（LC3）, p62, total- mammalian target of rapamycin （t- mTor）, phospho- mammalian target of rapamyein （p- mTor） and Beclin 1 were detected by Western blotting. （3） NRK-49F cells were cultured with 10 μg/L TGF-β1 for different times （0, 1, 4 h） in vitro after cultured with serum- free medium for 2.5 hours. The protein expressions of LC3 and p62 in NRK-49F cells were measured by Western blotting. Results （1） The morphology of NRK-49F cells changed from stellate to spindle shape after cultured with TGF- β1. The expressions of cell activation markers α- SMA and Col I gradually increased as the extend of stimulation time （all P 〈 0.05）. （2）TGF-β1 transiently increased the expressions of autophagy proteins p62 （peak value appeared after 4 h） and p- roTor （peak value appeared after 30 min）, while decreased Beclinl expression level （all P 〈 0.05）. （3） TGF-β1 decreased the protein expression of LC3- II in NRK-49F cells cultured with serum- free medium, whereas increased the protein expression of p62 at the same time （all P 〈 0.05）. Conclusions The autophagy activity of renal fibroblasts is inhibited by the TGF-β1 -induced cellular activation in vitro, which may contribute to the progression of renal interstitial fibrosis.
出处
《中华肾脏病杂志》
CSCD
北大核心
2017年第11期843-848,共6页
Chinese Journal of Nephrology
关键词
转化生长因子-Β1
纤维化
成纤维细胞
自噬相关蛋白
Transforming growth factor beta1
Fibrosis
Fibroblasts
Autophagy and its correlated proteins
