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MCP-1诱导的肾小管上皮-间充质细胞转化与整合素连接激酶表达变化的关系
引用本文:赵班,李慧凛,连耀国,吴华,郑法雷.MCP-1诱导的肾小管上皮-间充质细胞转化与整合素连接激酶表达变化的关系[J].北京医学,2009,31(3):150-154.
作者姓名:赵班  李慧凛  连耀国  吴华  郑法雷
作者单位:1. 卫生部北京医院肾内科,100730
2. 中国医学科学院,北京协和医院肾内科
摘    要:目的探讨单核细胞趋化蛋白-1(MCP-1)诱导体外培养的肾小管上皮细胞转分化的作用及其与细胞整合素连接激酶(ILK)表达变化的关系。方法体外培养的HKC,以未处理的HKC为阴性对照,以8ng/mlTGF-81处理的HKC为阳性对照,以不同浓度的MCP-1(0.1、1.0、10、100ng/m1)处理细胞,或以同一浓度的MCP-1(1ng/ml)在不同时间点(0h、12h、24h、36h、48h、72h)处理细胞。用半定量RT-PCR方法测定各组HKC细胞α—SMA mRNA的表达,Western blot方法测定各组HKC细胞α-SMA及ILK表达。在上述实验基础上进一步观察MEK抑制剂(PD98059,10μmol/L),p38MAPK抑制剂(SB203580,5/μmol/L)和ROCK抑制剂(Y27632,500μmol/L)对HKC细胞ILK表达的影响。结果MCP-1(0.1、1.0ng/m1)作用后HKC细胞α-SMA mRNA和α—SMA、ILK蛋白表达显著高于阴性对照组(P<0.01),但低于阳性对照组(P<0.01);其中以1.0ng/ml MCP-1组作用后α—SMAmRNA和α—SMA、ILK蛋白表达水平增高更为显著(P<1.001)。1.0ng/ml MCP-1作用后细胞α-SMA mRNA和α-SMA、ILK蛋白表达在0-48h内逐渐增加,48h达最高峰(P<0.01),72h时呈下降趋势。分别加入阻断剂PD98059、SB203580和Y27632后,HKC细胞ILK蛋白表达水平与MCP-1单独作用无显著性差异(P>0.05)。结论MCP-1可诱导肾小管上皮细胞转分化并呈时间浓度依赖性,该作用可能与MCP-1上调ILK的表达有关;本研究未发现MCP-1上调ILK的信号传导途径与MEK1、p38MAPK、ROCK途径有关。

关 键 词:肾小管上皮-间充质细胞转化  转化生长因子-β1  α-平滑肌肌动蛋白  单核细胞趋化因子-1  整合素连接激酶

Study in altered expression of integrin-linked kinase in Epithelial-mesenchymal transition of HKC induced by MCP-1
Institution:ZHAO Ban, LI Hui-lin , LIAN Yao-guo , et al (Department of Nephrology, Peking Union Medical College Hospital,Beijing 100730)
Abstract:Objective To explore the role of integrin-linked kinase (ILK) in MCP-1 induced epithelial to mesenchymal transition (EMT) of tubular epithelial cells. Methods Cultured human kidney tubular epithelial ceils (HKC) were divided into three groups,including negative control (untreated HKC),positive control (TGF-β1 8ng/ml treated HKC) and MCP-1 (0.1,1.0,10,100 ng/ml) treated HKC. HKC cells were incubated with the same concentration of MCP-1 (1.0 ng/ml) for various periods (0h, 12h, 24h, 36h, 48h, 72h). The effect of various chemical inhibitors,including PD98059 (10μmol/L) ,SB203580 (5μmol/L), Y27632 (500μmol/L), on ILK was also tested respectively, followed by incubating in absence of MCP-1 (1 ng/ml) for 24 hours. MCP-1 induced a-smooth muscle actin (α-SAM) mRNA expression was assessed by RT-PCR. MCP-1 induced α-SMA and ILK protein expressions were assessed by Western blot. Results MCP-1 induced α-SMA mRNA expression,α-SMA and ILK protein expressions increased higher than the negative control (P〈0.01) but lower than the positive control (P〈0.01) ,this increasement was in a time and dose-dependent manner, and reached the peak at 48h after MCP-1 treatment. The various chemical inhibitors or vehicle did not affect ILK expression induced by MCP-1 (1.0ng/ml). ILK expressions of cells did not changed when PD98059.SB203580 or Y27632 were added in the culture. Conclusion MCP-1 may induce EMT of cultured renal epithelial cells and this effect is closely related to up-regulation of ILK. However,MCP-1 induced ILK change is not related to MEK,p38 MAPK or ROCK pathway.
Keywords:Epithelial mesenchymal transition (EMT) Transforming growth factor-β1 (TGF-β1)α-Smooth muscle actin(α-SMA) Monocyte chemoattractant protein-1(MCP-1) Integrin-linked kinase(ILK)
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