摘要
背景心脏肥大细胞的糜酶参与心肌纤维化,但其作用机制尚不清楚。目的探讨糜酶对大鼠心脏成纤维细胞(CFs)胶原合成的影响及其与转化生长因子β1(TGF-β1)/Smad信号通路的关系。方法用胰酶消化法分离、培养新生SD大鼠的CFs,采用3H脯氨酸掺入法测定CFs的胶原合成,Western blot检测TGF-β1、磷酸化Smad2/3(P-Smad2/3)及总的Smad2/3的蛋白表达水平。结果糜酶以浓度依赖方式增加CFs的3H脯氨酸掺入率。15、30和60μg/L组3H脯氨酸掺入率分别为(520±75)、(684±62)和(769±58)计数/(min.孔),均高于对照组[(435±60)计数/(min.孔),P<0.05或P<0.01]。在30μg/L糜酶作用下,TGF-β1及P-Smad2/3蛋白表达水平呈时间依赖性改变,3、6和12h组均高于对照组(P<0.05或P<0.01);而Smad2/3蛋白表达水平与对照组比较,差异无统计学意义(P>0.05)。TGF-β1中和抗体和P-Smad2/3抑制剂预处理组3H脯氨酸掺入率均低于30μg/L糜酶组(P<0.01)。结论糜酶具有促进CFs胶原合成的作用,其机制可能与TGF-β1/Smad信号通路的活化有关。
Background Cardiac mast cell-derived chymase is involved in myocardial fibrosis, but the underlying mechanisms remain unclear. Objective To investigate the effect of chymase on the collagen synthesis and its relationship with transforming growth factor-β1（TGF-β1）/Smad pathway in rat cardiac fibroblasts （CFs ）. Methods CFs, from neonatal SD rats, were isolated by trypsinization. The collagen synthesis of CFs was determined by ^3 H-proline incorporation. The protein expressions of TGF-β1, phosphorylated Smad2/3 （P-Smad2/3） and total Smad2/3 were determined by immunoblotting in CFs. Results Chymase （15, 30 and 60 μg/L） increased the ^3 H-proline incorporation in a concentration-dependent manner. 30 μg/L chymase stimulation increased the pro- tein expressions of TGF-β1and P-Smad2/3 in a time-dependently, while little effect on Smad2/3 protein expression was found. The stimulatory effect of chymase on ^3 H-proline incorporation elicited by 30 μg/L chymase was blocked in the presence of TGF-β1antibody or staurosporine, a P-Smad2/3 inhibitor. Conclusion Chymase promotes collagen synthesis of rat CFs, TGF-β1Smad might be involved into the signal pathway.
出处
《中华高血压杂志》
CAS
CSCD
北大核心
2009年第8期743-747,共5页
Chinese Journal of Hypertension
基金
陕西省科学技术研究发展计划项目2005K13-G1-3
