摘要
目的观察转化生长因子-β1(TGF-β1)对大鼠心房、心室成纤维细胞胶原蛋白表达的不同影响,并探讨其可能的分子机制。方法用组织贴块法培养大鼠原代心房、心室成纤维细胞,经SABC免疫细胞化学染色方法鉴定细胞,分别进行以下实验:1应用羟脯氨酸消化法观察不同浓度TGF-β1(0、5、10ng/ml)及不同作用时间(6、12、24、48h)对心房、心室成纤维细胞羟脯氨酸含量的影响。2用筛选的TGF-β1最佳干预浓度及作用时间分别刺激心房、心室成纤维细胞,Western blotting检测α-平滑肌肌动蛋白(α-SMA)鉴定肌成纤维细胞后,采用羟脯氨酸法比较两种细胞培养液中羟脯氨酸的表达情况,RT-PCR检测二者Ⅰ、Ⅲ型胶原蛋白的m RNA表达,Western blotting检测二者Smad2/3、p-Smad2/3及Smad7蛋白的表达情况。结果 TGF-β1对大鼠心房、心室成纤维细胞均具有促进胶原蛋白合成的作用,当TGF-β1刺激条件为5ng/ml、24h时作用最强。用上述条件TGF-β1刺激心房、心室成纤维细胞,与对照组相比,心房成纤维细胞Ⅰ、Ⅲ型胶原蛋白,p-Smad2/3表达增加,Smad7表达降低,差异有统计学意义(P<0.05);心房成纤维细胞TGF-β1刺激组与心室成纤维细胞TGF-β1刺激组相比,两组Smad2/3的表达差异无统计学意义,但心房成纤维细胞TGF-β1刺激组Ⅰ、Ⅲ型胶原蛋白,p-Smad2/3的表达明显升高,Smad7的表达明显降低(P<0.05)。结论 TGF-β1能诱导心脏成纤维细胞转化为肌成纤维细胞,可引起细胞胶原代谢异常,而且心房成纤维细胞中胶原代谢异常表现较心室成纤维细胞明显,其机制可能与TGF-β1/SMAD信号通路相关。
Objective To obser ve the effect of transforming grow th factor beta 1（TGF-β1） on the ex pression of collagen in rat atrial and ventricular fibroblasts, and to investigate its specific molecular mechanisms. Methods Tissue explant attachment was used to culture fibroblasts obtained from the atrium and ventricle of rat heart, and they were identified with SABC immunocytochemical staining, and then the following experiments were carried out.（1） Hydroxyproline digestion was performed to study the effects of TGF-β1, within different concentrations（0, 5, 10ng/ml） and different action time（6, 12, 24, 48h） on the content of hydroxyproline in rat＇s atrial and ventricular fibroblasts.（2） Rat＇s atrial and ventricular fibroblasts were stimulated with TGF-β1 in optimal concentration and action time, the expression of α-smooth muscle actin（α-SMA） was determined with Western blotting, and the expressions of typeⅠ and Ⅲ collagen m RNA were evaluated with reverse-transcription PCR. The contents of hydroxyproline in the respective cells were measured with hydroxyproline determination. Western blotting was used to measure the protein expression of Smad2/3, p-Smad2/3 and Smad7. Results（1） TGF-β1 was shown to stimulate the collagen synthesis in rat＇s atrial and ventricular fibroblasts, and the optimal stimulus was TGF-β1 concentration 5ng/ml with action time of 24 h.（2） After being stimulated by optimal stimulation effect of TGF-β1, the expression of typeⅠ and Ⅲ collagen and p-Smad2/3 increased, while that of Smad7 decreased significantly only in atrial fibroblasts（P〈0.05）, but not in ventricular fibroblasts. No statistical difference was found in the expression of Smad2/3 between the atrial and ventricular fibroblasts after being stimulated by TGF-β1under optimal stimulating conditions. Conclusion TGF-1 can induce dysbolism of collagen of cardiac fibroblasts with abnormal expression of cytoskeletal protein, which may occur more obviously in rat＇s atrial fibroblasts than
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2015年第7期540-546,共7页
Medical Journal of Chinese People's Liberation Army
基金
国家自然科学基金面上项目(81300140)~~
