缺氧不同时间对人胚肺成纤维细胞RHO/RHO激酶信号通路及CTGF表达的影响  

The Effect of Hypoxia on the Expression of RHO/RHO Signaling Pathway and CTGF of Human Embryonic Fibroblasts

作　　者：高艳[1] 谢敏[1] 何正平[1] 张旭[1] 郑家群[1] 

机构地区：[1]四川大学华西医院呼吸内科,成都610041

出　　处：《四川大学学报（医学版）》2010年第6期946-950,共5页Journal of Sichuan University(Medical Sciences)

基　　金：四川省科技厅攻关项目(项目号:0040205301469)资助

摘　　要：目的观察不同时点低氧条件下,对人胚肺成纤维细胞(MRC-5)RHO/RHO激酶信号通路、结缔组织生长因子(CTGF)表达及Ⅰ型胶原(ColⅠ)合成的影响并探讨其可能机制。方法以MRC-5为研究对象,在常氧和缺氧(37℃、5%CO2、2%O2、93%N2)条件下分别孵育MRC-5细胞0、1.5、3、6、12、24、48、60h,用Westernblot法检测低氧诱导因子-1α(HIF-1α)、RhoA、ROCK1、CTGF蛋白的表达;RT-PCR技术检测HIF-1α、ROCK1、CTGF及ColⅠmRNA的表达;ELISA法测定细胞上清ColⅠ的表达。结果①体外培养的MRC-5细胞在常氧条件下表达基础水平的RhoA、ROCK1、CTGF,在低氧条件下RhoA、ROCK1、CTGF的蛋白表达水平增强(P<0.01),具有时间依赖性,以60h表达最高,且在低氧条件下,不同时点MRC-5细胞的RhoA、ROCK1蛋白表达水平与CTGF蛋白表达水平呈正相关关系。②与常氧对照组比较,低氧作用1.5h后即出现ROCK1、CTGF、ColⅠmRNA的表达上调(P<0.01),并随着低氧作用时间延长而逐渐增强,以60h表达最高;③与常氧对照组比,在低氧条件下,不同时点MRC-5细胞分泌ColⅠ均增加,以24h最高。结论缺氧不同时间均可上调细胞CTGF在mRNA和蛋白水平的表达、增加ColⅠ的合成,其机制可能部分是通过激活RHO/RHO激酶信号通路及上调HIF-1α的表达促发CTGF的高表达进而引发肺成纤维细胞细胞外基质表达增加等一系列促纤维化反应。Objective 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. Methods Human embryonic fibroblasts were cultured under hypoxic condition （37 ℃,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-1α, RhoA, ROCKland CTGF were assayed by Western blot. RT-PCR was performed to detect the mRNA levels of HIF-1α, ROCK1, CTGF and Col Ⅰ. The concentration of collagen type Ⅰ （Col Ⅰ ） in fibroblast cells supernatant were determined by ELISA. Results 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. ②After 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 Co1 Ⅰ in fibroblast cells supernatant increased in different time points of hypoxia and peaked after 24 h of hypoxia. Conclusion Different time points of hypoxia all signi candy up-regulated the expression of protein and mRNA of CTGF and increased the secretion of Col Ⅰ. Hypoxia could represent a potential brotic stimulus possiblely through activation of the RHO/ RHO signaling pathway, up-regulating the expression of HIF-1α and CTGF, triggering a series of brotie responses.

关 键 词：低氧 人胚肺成纤维细胞 低氧诱导因子-1Α RHOA ROCK1 结缔组织生长因子 

分 类 号：R563[医药卫生—呼吸系统]