机构地区:[1]北京大学第一医院北京大学肾脏病研究所,100034
出 处:《中华医学杂志》2003年第13期1161-1165,共5页National Medical Journal of China
基 金:国家自然科学基金 (3 0 0 70 3 5 1);教育部跨世纪优秀人才基金 (3 9910 2 10 474 2 3 1 C0 4)资助项目
摘 要:目的 探讨 p38MAPK信号转导途径对IL 1β诱导肾小管上皮细胞转分化的影响及其导致的功能改变。方法 以培养的人近端肾小管上皮细胞 (HK 2 )为研究对象 ,给予IL 1β(10ng/ml)刺激 2 4h ,在阻断实验中加入SB2 0 35 80阻断 p38通路。采用蛋白印记杂交法检测 p38MAPK的磷酸化程度 ;细胞表型特征检测包括细胞形态变化、标志蛋白 (角蛋白、α SMA)的表达及分布、细胞增殖、黏附和移行能力。结果 (1)IL 1β刺激 6~ 4 8h可使α SMA表达上调 ,刺激 2 4h可使角蛋白表达减弱、α SMA分布紊乱 ,但细胞形态变化不明显 ;(2 )IL 1β在 5min时可使p38激活达高峰 ,较基础水平升高 2~ 3倍 (P <0 0 5 ) ;至 12 0min时 p38则呈持续激活状态 ;(3)p38阻断剂 (SB2 0 35 80 )对HK 2细胞α SMA的基础表达有抑制作用 ,抑制率 37 13% (P <0 0 1) ;同时可明显抑制IL 1β刺激的α SMA表达 ,抑制率 4 7 0 7% (P <0 0 0 1) ;(4)IL 1β及 p38阻断剂并不影响HK 2细胞之间的黏附能力 ,但IL 1β可使细胞的移行能力增强 (P <0 0 1) ,而 p38阻断剂可明显抑制其移行能力。IL 1β并不影响HK 2细胞增殖。结论 IL 1β可以激活肾小管上皮细胞p38/MAPK ,并可使其发生表型转化 ,这一作用部分通过 p38信号通路所介导。Objective To investigate the role of p38MAPK signaling pathway in interleukin (IL)-1β induced transdifferentiation and its functional influences in human renal proximal tubular cell line HK-2 cells.Methods Human renal proximal tubular cells, cell line HK-2, were cultured and then co-incubated with IL-1β(10 ng/ml)for 24 hours.The expression and distribution of cytokeratin and α-smooth muscle actin (SMA), markers of transdifferentiation of renal proximal tubular cells, were detected by immunofluorescence staining and confocal microscopy.Western blot technique was used to detect the expression of α-SMA 2,4, 6, 12, 24, and 48 hours after IL-1β stimulation.The morphology of cells was monitored from the 1st to the 5th day.Expression of α-SMA, phosphorylation of p38MAPK was assayed by western blot.Specific p38MAPK inhibitor SB203580 was added into the culture of HK-2 cells, 24 hours later IL-1β was added for 24 hours Cell-cell adhesion and migration assay were performed.Inverted microscopy was used to examine the morphology of cells after stimulation of IL-1β for 24 hours.Western blot technique was used to detected total and phosphorylated p38MAPK after stimulation of IL-1β for 2?5?15?30?60 and 120 min.Results The phosphorylation of p38MAPK was increased after treatment of IL-1β, and reached the level of 1.7 times the basic level 5~30 minutes after stimulation ( P <0.05).Almost no expression of α-SMA was shown in the control group.Two hours after IL-1β stimulation, the expression of α-SMA was increased a little followed by an up-regulated expression of α-SMA 6 to 48 hours after the stimulation.The expression of α-SMA was down-regulated by 40% in the SB203580 group in comparison with that in the control ( P <0.01) and by 50% in comparison with that in the IL-1β group ( P <0.001).The cell number was not significantly different between the IL-1β group and control group ( P >0.05). A decreased expression of cytokeratin and disordered distribution of α-SMA were shown in the cells 24 hours after stimulat
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