雷帕霉素抑制结缔组织生长因子诱导的成肌纤维细胞增殖及细胞外基质蛋白的分泌  

Inhibitory effect of rapamycin on connective tissue growth factor-stimulated cell proliferation and fibronectin secretion in myofibroblasts

作　　者：高绪霞[1] 黄海长[1] 李晓玫[1] 

机构地区：[1]北京大学第一医院肾脏内科北京大学肾脏病研究所,100034

出　　处：《中华肾脏病杂志》2009年第9期678-682,共5页Chinese Journal of Nephrology

基　　金：基金项目：国家自然科学基金（30570851）;教育部新世纪优秀人才基金（985-2-071-113）

摘　　要：目的探讨雷帕霉素（RAPA）对于结缔组织生长因子（CTGF）生物学作用的影响及可能机制。方法分别用RAPA20μg／L和40μg／L预处理成肌纤维细胞（MyoF）30min后再加入CTGF100μg／L，与单独加入CTGF（CTGF组）和未加任何刺激的MyoF（对照组）进行比较。采用BrdU掺入法检测细胞的增殖反应；用Western印迹法检测细胞上清中纤连蛋白（FN）的水平及细胞ERK1／2信号的磷酸化。结果与对照组相比，CTGF（100μg／L）显著促进MyoF增殖（P〈0．01），增加上清中FN的蛋白水平（P〈0．05）。与CTGF组相比，RAPA20μg／L及40μg／L预处理可使细胞增殖率显著下降，分别下降了62％和70％（均P〈0．05），但两个剂量之间差异无统计学意义；使细胞上清中FN的蛋白水平分别下降了15％和44％，后者与CTGF组的差异有统计学意义（P〈0．05）。CTGF（100μg／L）刺激10min可致MyoF的ERK1／2发生磷酸化，RAPA40μg／L预处理细胞30min可显著降低CTGF诱导的ERK1／2磷酸化。以ERK1／2活化特异抑制剂PD98059（50μmol／L）预处理30min后，可以抑制CTGF诱导的细胞增殖效应（7％±5％比85％±7％，P〈0．01）和FN的分泌效应（1．0±0．1比1．6±0．3，P〈0．05）。结论RAPA具有部分抑制CTGF的促增殖和促细胞外基质分泌的作用，其作用可能通过抑制ERK1／2信号通路实现。Objective To investigate the inhibitory effect and associated mechanism of rapamycin on proliferation and extracellular matrix （ECM） secretion in myofibroblasts stimulated by connective tissue growth factor （CTGF）. Methods Primary cultivated myofibroblasts were divided into 6 groups： control, CTGF （100 μg/L）, rapamycin 20 μg/L＋CTGF 100 μg/L, rapamycin 40 μg/L ＋CTGF 100 μg/L, rapamycin 20 μg/L, and rapamycin 40 μg/L alone. 5′- bromodeoxyuridine （BrdU） incorporation assay was used to detect the myofibroblast proliferation. Western blot was used to analysis the secretory FN protein in the supernatant medium of cultured myofibroblasts and the ERK1/2 phosphorylation in myofibroblasts. Results CTGF （100 μg/L） incubation significantly increased the number of Brdu positive myofibroblasts（P〈0.01 ） and the level of FN protein secretory （P〈0.05） in cell supernatant medium compared with control group, respectively. The number of Brdu positive myofibroblasts markedly decreased by 62% and 70% （P 〈0.05） in rapamycin 20 μg/L＋CTGF 100 μg/L and rapamycin 40 μg/L＋CTGF 100 μg/L groups, respectively. The FN protein levels in supernatant were decreased by 15% and 44% compared with CTGF 100 μg/L group, respectively; but the difference of FN protein levels was significant only in rapamycin 40 μg/L group （P〈0.05）. CTGF could activate ERK1/2 at 10 minutes; but as myofibroblasts were pretreated with rapamycin 40 μg/L for 30 min, it abolished CTGF-induced ERK1/2 phosphoralation. PD98059, the specific inhibitor of ERK1/2, could block the effect of CTGF-induced proliferation （7%±5% vs 85%±7%, P〈0.01） and FN secretion （1.0±0.1 vs 1.6± 0.3, P〈0.05）. Conclusions Rapamycin partially suppresses the proliferation and ECM secretion of myofibroblasts induced by CTGF. Its effect may be through inhibiting CTGF-induced activation of ERK1/2 signaling pathway.

关 键 词：细胞增殖 纤连蛋白类 西罗莫司 成肌纤维细胞 结缔组织生长因子 ERK1/2信号通路 

分 类 号：R285.5[医药卫生—中药学]