缺氧对肺成纤维细胞表达结缔组织生长因子的影响

Effects of hypoxia on the expression of connective tissue growth factor in lung fibroblast

目的观察不同缺氧时间对肺成纤维细胞结缔组织生长因子(CTGF)表达的影响。方法原代培养的人肺成纤维细胞在缺氧(37℃、5%CO2、5%O2、90%N2)条件下分别孵育0、1.5、3、6及12 h,同时用抗CTGF抗体干预的人肺成纤维细胞在缺氧条件下培养12 h。RT-PCR法测定细胞CTGF mRNA、基质金属蛋白酶9(MMP9)mRNA表达情况,ELISA法测定细胞培养上清液CTGF、MMP9的浓度。结果缺氧刺激肺成纤维细胞CTGF mRNA高表达出现在1.5 h,并保持一相对平台至6 h,12 h开始下降。MMP9 mRNA在缺氧1.5 h出现高表达,并保持持续增高的趋势。ELISA结果证实CTGF与MMP9一样其峰浓度在缺氧12 h,用抗CTGF抗体干预的成纤维细胞组较未干预组表达MMP9量明显减少。结论缺氧可刺激人肺成纤维细胞高表达CTGF;缺氧可能是通过刺激成纤维细胞高表达CTGF,进而调节细胞分泌MMP9的量,参予细胞外基质沉积和气道重构。

Objective To observe the effects of hypoxia on the expression of connective tissue growth factor （CTGF） in lung fibroblast at different time points.Methods Human fibroblast cells were cultured under hypoxic condition for 0 h, 1.5 h, 3 h, 6 h and 12 h, respectively. The cells cultured in hypoxic condition for 12 h were treated by anti-CTGF antibodies. RT-PCR was performed to detect CTGF and matrix metalloproteinase 9 （MMP9） mRNA levels. The concentration of CTGF and MMP9 protein in fibroblast cells supernatant were detemained by ELISA. Results Hypoxia stimulation of fibroblast cells induced CTGF mRNA expression within 1.5 h, and the expression level remained at a plateau up to 6 h, then decreased by 12 h. The level of MMP9 mRNA increased significantly within 1.5 h with an elevated trend. ELISA revealed that both levels of CTGF and MMP9 protein/cell in medium conditioned by fibroblast cells exposed to hypoxia were maximal at 12 h. The level of MMP9 in the CTGF-Ab treated group were significantly decreased compared to untreated group. Conclusions These findings suggest that hypoxia stimulates fibroblast cells to release CTGF as a mitogen factor, which initiates the fibrosis cascade and airway remodeling by regulating the balance of extracellular matrix synthesis and degradation via MMP9 secreted by fibroblast cells in response to CTGF.