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观察细胞因子信号传导抑制蛋白1(suppressors of cytokine signaling,SOCS-1)对抑瘤素M(oncostatin M,OSM)诱导的肾小管上皮细胞转分化的影响。体外培养人肾近曲小管上皮细胞(HKC),应用脂质体2000分别转染pCR3.1/SOCS-1表达质粒和pCR3.1空质粒载体,G418筛选阳性克隆,应用OSM(10ng/mL)进行刺激。培养48h后收集细胞及上清液,分别采用Western蛋白印迹检测细胞角蛋白18(cytokeratin18,CK18)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、SOCS-1和磷酸化信号转导及转录激活因子1(phospho-signal transducers and activators of transcription,p-STAT1)的表达;采用酶联免疫吸附实验测定细胞上清液中Ⅰ型胶原(collagen I,Col I)和纤维连接蛋白(fibronectin,FN)的分泌;采用逆转录-聚合酶链反应检测CK18、α-SMA mRNA的表达。结果显示,与对照组相比,OSM组肾小管上皮细胞α-SMA及p-STAT1蛋白的合成增加,α-SMA mRNA表达增加,细胞培养上清液Col I和FN的含量增加,而CK18蛋白及mRNA的表达减少。SOCS-1过表达能抑制OSM刺激引起的α-SMA和p-STAT1的表达,减少Col I和FN的分泌,下调α-SMA mRNA的表达,同时能够逆转OSM刺激引起的CK18蛋白及mRNA的表达。由此可见,SOCS-1过表达能抑制OSM诱导的肾小管上皮细胞转分化,此过程可能与STAT1的磷酸化受抑有关。
To investigate the effects of SOCS-1 on the transdifferentiation of renal tubular epithelial cells induced by oncostatin M (OSM). Human renal proximal tubule epithelial cells (HKC) were cultured in vitro. The pCR3.1 / SOCS-1 expression plasmid and the pCR3.1 empty plasmid vector were transfected with Lipofectamine 2000. The positive clones were screened by G418. OSM (10ng / mL) To stimulate. After cultured for 48h, the cells and supernatant were collected. Western blotting was used to detect the expression of cytokeratin 18 (CK18), α-smooth muscle actin (α-SMA), SOCS-1 and phosphorylation The expression of p-STAT1 and phospho-signal transducers and activators of transcription 1 were detected by enzyme-linked immunosorbent assay (ELISA). Collagen I (Col I) and fibronectin Fibronectin (FN) secretion. The expression of CK18 and α-SMA mRNA was detected by reverse transcription-polymerase chain reaction. The results showed that compared with the control group, the synthesis of α-SMA and p-STAT1 protein in renal tubular epithelial cells of OSM group increased, the expression of α-SMA mRNA increased, the contents of Col I and FN in the cell culture supernatant increased, while the CK18 protein And mRNA expression decreased. Overexpression of SOCS-1 could inhibit the expression of α-SMA and p-STAT1 induced by OSM stimulation, decrease the secretion of Col I and FN and down-regulate the expression of α-SMA mRNA, and also reverse the expression of CK18 protein and mRNA induced by OSM stimulation . Thus, overexpression of SOCS-1 can inhibit OSM-induced renal tubular epithelial cell transdifferentiation, which may be related to the inhibition of STAT1 phosphorylation.