低氧通过p38丝裂素激活蛋白激酶途径刺激人肾间质成纤维细胞表达结缔组织生长因子  

Hypoxia stimulates expression of connective tissue growth factor through p38 signaling pathway in human renal interstitial fibroblasts

作　　者：荣琳琳[1,2] 黄海长[1] 喻陆[3] 李惊子[1] 

机构地区：[1]北京大学第一医院肾内科,北京100034 [2]南方医科大学研究生四队 [3]中国人民解放军305医院肾内科,血液净化中心

出　　处：《北京大学学报（医学版）》2005年第4期378-381,共4页Journal of Peking University:Health Sciences

基　　金：国家自然科学基金(30270612);教育部教育振兴行动计划特殊专项("九八五"工程)资助~~

摘　　要：目的:研究低氧条件下人肾间质成纤维细胞表达结缔组织生长因子(connectivetissuegrowthfactor,CTGF)及其可能的信号途径,从而探讨低氧导致肾间质纤维化的分子机制。方法:以人肾间质成纤维细胞TK173作为研究对象,应用Westernblot技术,检测低氧标记蛋白分子,低氧诱导因子-1α(hypoxiainducedfactor-1α,HIF-1α)蛋白作为低氧标记物;比较低氧(1%O2,体积分数)和正常氧(21%O2,体积分数)条件下TK173细胞培养12,24,48h后CTGF蛋白水平;低氧刺激TK173细胞30min,1h,6h和12h,运用抗磷酸化抗体检测丝裂素激活蛋白激酶(MAPKs)活化;在低氧刺激半小时前分别加入p38、细胞外信号调节激酶(ERK1/2)、c-Jun-N末端蛋白激酶(JNK)信号通路特异阻断剂SB203580,PD98059,SP600125,低氧培养24h后检测细胞CTGF蛋白水平。用RT-PCR技术分析CTGFmRNA水平变化。结果:TK173细胞正常氧组仅有HIF-1α蛋白微弱表达,低氧组有高水平HIF-1α蛋白表达。在低氧条件下培养12h后细胞CTGF蛋白增加,24h时CTGF蛋白表达最强,是正常氧组的(2.1±0·1)倍,至48h降至对正常氧组水平;同时低氧培养刺激CTGFmRNA表达,在1h时开始增加,6h时达高峰为正常氧组的(6.6±1·0)倍,24h后恢复基础水平。低氧可以活化MAPKs,JNK在30min达到高峰,ERK1/2、p38在刺激1h时达高峰,6h减弱,12h减至基础水平;应用ERK1/2抑制剂PD98059和JNK抑制剂SP00125不能减弱低氧刺激CTGF蛋白表达增加的效应,但应用P38活化抑制剂SB203580可以明显减少低氧刺激的CTGF蛋白和mRNA的增加。结论:低氧可刺激人肾间质成纤维细胞表达CTGF,并且该作用依赖p38通路的活化。Objective ： To assess the expression of connective tissue growth factor （ CTGF）, and relevant mechanism for the regulation of CTGF expression by hypoxia in human renal interstitial fibroblast. Methods： A human renal interstitial fibroblast cell line TK173 was treated under hypoxia （1% O2） or nomoxia （21%O2） condition. The expressions of HIF1-α, hypoxia marker protein, and CTGF protein were analyzed by Western blotting. PT-PCR was carried out to measure the levels of CTGF mRNA. The activations of MAPKs （ERK, JNK, p38） signaling pathways were assessed at different time points （30 min, 1 h, 6 h, and 12 h ） , and the changes of CTGF expression were detected after the inhibitors of activation of MAPKs were applied, respectively. Results： The expression of HIF-1α protein appeared in cells under hypoxia for 6 h. The expressions of CTGF protein were up-regulated in TK173 cells under hypoxia for 12 h, reached the peak levels in 2 folds of normoxia group cells for 24 h, and return to the levels of control cells by 48 h. The levels of CTGF mRNA were elevated in cells under hypoxia for 1 h, significantly increased at 6 h （6.6 ± 1.0, P = 0. 000 2） , and returned to the levels of normoxia group cells by 24 h. Activations of ERK1/2, JNK and p38 were seen in hypoxic cells. Activation of ERK1/2 and JNK were occurred as early as at l0 rain, and reached the peak levels at 1 h, while the peak levels of activated JNK were seen at 30 rain, then the levels of activated ERK1/2, p38, and JNK were all declined at 6 h, back to the baseline levels at 12 h. Blockade of ERK activation with PD98059. and blockade of JNK activation with SP600125 did not suppress hypoxia-induced expression of CTGF protein, whereas blockade of p38 MARK activation with SB203580 abolished hypoxia-induced expressions of CTGF protein and CTGF mRNA. Conclusion： Hypoxia could stimulate the expression of CTGF in human renal interstitial fibroblast through the activation of p38 MARK signaling pathway.

关 键 词：有丝分裂素激活蛋白激酶类 成纤维细胞 结缔组织生长因子 肾 

分 类 号：R692.32[医药卫生—泌尿科学]