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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 Chymase in cardiac mast cells is involved in myocardial fibrosis, but its mechanism of action is unclear. Objective To investigate the effect of chymase on collagen synthesis in rat cardiac fibroblasts (CFs) and its relationship with TGF-β1 / Smad signaling pathway. Methods The CFs of newborn SD rats were isolated and cultured by trypsin digestion. The collagen synthesis of CFs was determined by 3H proline incorporation method. The expressions of TGF-β1, P-Smad2 / 3 and Total Smad2 / 3 protein expression levels. As a result, chymase increased 3H proline incorporation of CFs in a concentration-dependent manner. The 3H-proline incorporation rates in the 15, 30 and 60 μg / L groups were (520 ± 75), (684 ± 62) and (769 ± 58) counts / ± 60) counts / (min. Wells), P <0.05 or P <0.01]. The expression of TGF-β1 and P-Smad2 / 3 in 30μg / ml chymase was time-dependently changed in 3, 6 and 12h groups (P <0.05 or P <0.01), while Smad2 / 3 protein expression levels compared with the control group, the difference was not statistically significant (P> 0.05). The 3H-proline incorporation of TGF-β1 neutralizing antibody and P-Smad2 / 3 inhibitor pretreatment group were lower than that of 30μg / L chymase group (P <0.01). Conclusion Chymase can promote the collagen synthesis of CFs, which may be related to the activation of TGF-β1 / Smad signaling pathway.