Gao Yan, Xie Min, He Zheng-ping, Zhang Xu, Zheng Jia-qun
Department of Respiratory, West China Hospital, Sichuan University, Chengdu 610041, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2010 Nov;41(6):946-50.
To observe the effects of hypoxia on cell proliferation and the expression of RHO/RHO signaling pathway, connective tissue growth factor (CTGF) and the collagen type I (Col I) in human embryonic fibroblasts at different time points, and study the possible mechanism of hypoxia-induced pulmonary fibrosis.
Human embryonic fibroblasts were cultured under hypoxic condition (37 degrees C, 5 CO2, 2% O2, 93% N2) for 0 h, 1.5 h, 3 h, 6 h, 12 h, 24 h, 48 h and 60 h respectively. The protein levels of HIF-1alpha, RhoA, ROCKland CTGF were assayed by Western blot. RT-PCR was performed to detect the mRNA levels of HIF-1alpha, ROCK1, CTGF and Col I. The concentration of collagen type I (Col I) in fibroblast cells supernatant were determined by ELISA.
(1) Basal levels of RhoA, ROCK1, CTGF protein of MRC-5 were observed in normoxia, but the protein levels of RhoA, ROCK1 and CTGF were up-regulated after 1.5 h of hypoxia and increased further with longer exposure to hypoxia. It also demonstrated significant positive correlation between the protein levels of RhoA, ROCK1 and CTGF at different time points of hypoxia. (2) After 1.5 h, mRNA expression of ROCK1, CTGF and Col I was elevated and increased further with longer exposure to hypoxia and peaked after 60 h of hypoxia compared to normoxia. (3) Compared with normoxia, the concentration of Col I in fibroblast cells supernatant increased in different time points of hypoxia and peaked after 24 h of hypoxia.
Different time points of hypoxia all significantly up-regulated the expression of protein and mRNA of CTGF and increased the secretion of Col I. Hypoxia could represent a potential brotic stimulus possibly through activation of the RHO/RHO signaling pathway, up-regulating the expression of HIF-1alpha and CTGF, triggering a series of brotic responses.
观察缺氧对人胚肺成纤维细胞在不同时间点细胞增殖及RHO/RHO信号通路、结缔组织生长因子（CTGF）和Ⅰ型胶原（Col Ⅰ）表达的影响，探讨缺氧诱导肺纤维化的可能机制。
将人胚肺成纤维细胞分别在缺氧条件（37℃、5% CO₂、2% O₂、93% N₂）下培养0 h、1.5 h、3 h、6 h、12 h、24 h、48 h和60 h。采用蛋白质免疫印迹法检测缺氧诱导因子-1α（HIF-1α）、RhoA、Rho相关卷曲螺旋蛋白激酶（ROCK）及CTGF的蛋白水平。采用逆转录聚合酶链反应（RT-PCR）检测HIF-1α、ROCK1、CTGF及Col Ⅰ的mRNA水平。采用酶联免疫吸附测定法（ELISA）检测成纤维细胞培养上清液中Ⅰ型胶原（Col Ⅰ）的浓度。
（1）常氧下可观察到MRC-5细胞中RhoA、ROCK1、CTGF蛋白的基础水平，但缺氧1.5 h后RhoA、ROCK1和CTGF蛋白水平上调，且随着缺氧时间延长进一步升高。不同缺氧时间点RhoA、ROCK1和CTGF蛋白水平之间呈显著正相关。（2）缺氧1.5 h后，ROCK1、CTGF和Col Ⅰ的mRNA表达升高，且随着缺氧时间延长进一步增加，与常氧相比，缺氧60 h时达到峰值。（3）与常氧相比，缺氧不同时间点成纤维细胞培养上清液中Col Ⅰ浓度升高，缺氧24 h时达到峰值。
不同时间点的缺氧均显著上调CTGF的蛋白和mRNA表达，增加Col Ⅰ的分泌。缺氧可能通过激活RHO/RHO信号通路，上调HIF-1α和CTGF的表达，引发一系列纤维化反应，从而成为潜在的纤维化刺激因素。
