缺氧对肺成纤维细胞表达结缔组织生长因子和基质金属蛋白酶-9的影响

Effect 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)和基质金属蛋白酶9(MMP-9)表达的影响。方法原代培养的人肺成纤维细胞在缺氧(37℃,5%CO_2,5%O_2,90%N_2)条件下分别孵育0、1.5、3、6、12h,反转录聚合酶链反应(RT-PCR)法测定细胞CTGF mRNA、MMP-9 mRNA表达情况,并以酶联免疫吸附试验(ELISA)法测定培养细胞上清液CTGF、MMP-9浓度。结果缺氧刺激成纤维细胞CTGF mRNA高表达出现在1.5h,并保持一相对平台至6h,12h开始下降。MMP-9 mRNA在缺氧1.5h出现高表达,并保持一持续增高的趋势。ELISA结果证实CTGF与MMP-9一样其峰浓度在缺氧12 h。用抗CTGF抗体干预的成纤维细胞组较未干预组表达MMP-9量明显减少。结论缺氧可促使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MMP-9的量,来达到促进细胞外基质沉积、气道重塑作用而形成纤维化。

Objective To observe the effects of hypoxia on the expression of connective tissue growth factor （CTGF） and matrix metalloproteinase 9 （MMP-9） in lung fibroblasts at different time points. Methods Fibroblasts were cultured at hypoxic condition 0, 1.5, 3, 6, 12 h respectively. CTGF mRNA and MMP-9 mRNA level were detected in each group by reverse transcription-polymerase chain reaction （RT-PCR）. The concentration of CTGF and MMP-9 protein in fibroblasts supernatant were determined using enzyme-linked immunosorbent assay （ELISA）. Results Hypoxia stimulated fibroblasts increased the level of CTGF mRNA within 1.5 h, and the levels remained at a plateau up to 6 h, and then decreased by 12 h. The level of MMP-9 mRNA increased significantly within 3 h, and the levels kept the trend for increasing. ELISA revealed that the levels of both CTGF and MMP-9 protein/cell in medium conditioned by fibroblasts exposed to hypoxia were maximal at 12 h. The level of MMP in the CTGF-Ab treated groups was significantly decreased compared to the untreated groups. Conclusions These findings suggest that hypoxia stimulates fibroblasts 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 MMP-9 which is secreted by fibroblast cells in response to CTGF.