结缔组织生长因子通过ERK1／2途径刺激肾脏成肌纤维细胞增殖和Ⅰ型胶原生成

Connective tissue growth factor stimulates cell proliferation and collagen type Ⅰ secretion through ERK1/2 signaling pathway in cultured renal myofibroblasts

目的观察结缔组织生长因子（CTGF）对大鼠肾脏成肌纤维细胞增殖及细胞外基质（ECM）成分Ⅰ型胶原生成的影响，并探讨ERKI／2途径在其中的作用。方法原代培养肾皮质成肌纤维细胞，应用5’-溴-2’-脱氧尿嘧啶（Brdu）掺入法和细胞计数法检测细胞增殖；Western印迹法检测细胞上清液中Ⅰ型胶原的蛋白水平及细胞内ERK1／2的磷酸化水平。结果CTGF明显促进成肌纤维细胞的增殖，呈时间和剂量依赖性，100μg/L组第4天时，细胞数为对照组的1．6倍（P〈0．05）。CTGF刺激可显著增加培养上清液中Ⅰ型胶原的蛋白水平[为对照组的（2．6±1．2）倍，P〈0．05]。CTGF刺激细胞5min后ERK1／2即发生磷酸化，持续至15min，30min以后迅速降至基础水平。用ERK1／2阻断剂PD98059预处理30min后，CTGF促进细胞增殖效应（7％±5％比85％±7％，P〈0．01）和促Ⅰ型胶原分泌作用（1．0±0．1比1．6±0．3，P〈0．05）被显著抑制。结论CTGF能够促进成肌纤维细胞的增殖和Ⅰ型胶原的分泌，其作用可能通过ERK1／2信号通路实现。

Objective To examine the effects of connective tissue growth factor （CTGF） on cell proliferation and production of collagen type Ⅰ in cultured rat cortical myofibroblasts, and to investigate the role of ERK1/2 siganling pathway. Methods Myofibroblasts were obtained from normal rat renal cortex. 5′-bromodeoxyuridine （BrdU） incorporation assay and cell counting were used to detect cell proliferation. Western blot analysis was used to detect the levels of collagen type Ⅰ in the supernatant medium and the activation of ERK1/2 signaling pathway in cultured myofibroblasts. Results CTGF could induce the proliferation of myofibroblasts in a dose- and time-dependent manner. Cell number of 100 μg/L CTGF at day 4 was 1.6 folds of control group （P〈0.05）. Incubation with 100 μg/L CTGF also significantly increased secretion of collagen type I in the supernatant medium compared with control group （2.6±1.2 folds over control, P〈0.01）. ERK1/2 activation occurred as early as 5 minutes following 100 μg/L CTGF treatment, and persisted till 15 minutes later, and then declined back to the basal level after 30 minutes. Pretreatment with 50 μmol/L PD98059, a specific inhibitor of ERK1/2 pathway, abolished the effects of CTGF-induced cell proliferation （7%±5% vs 85%±7%, P〈0.01） and CTGF-increasedsecretion of collagen type Ⅰ （1.0±0.1 vs 1.6±0.3, P〈0.05）. Conclusion CTGF promotes cell proliferation and secretion of collagen type Ⅰ through ERK1/2 pathway in primary cultured rat myofibroblasts.