激活素受体样激酶1在系统性硬化病患者皮肤成纤维细胞中的表达及作用  

Expression of activin receptor-like kinases 1 in dermal fibroblasts from patients with systemic scleroderma and its roles

作　　者：赵信[1] 满孝勇[1] 李伟[1] 周炯[1] 陈佳琦[1] 蔡绥勍[1] 郑敏[1] 

机构地区：[1]浙江大学医学院附属第二医院皮肤科,杭州310009

出　　处：《中华皮肤科杂志》2012年第11期813-816,共4页Chinese Journal of Dermatology

基　　金：国家自然科学基金（81171497）

摘　　要：目的探讨激活素受体样激酶1（ALK1）在系统性硬化病患者皮肤成纤维细胞中的表达及其在纤连蛋白和I型纤溶酶原激活物抑制因子（PAI-1）产生中的作用。方法Western印迹和免疫荧光法观察比较ALK1在14例健康人和12例系统性硬化病患者皮肤成纤维细胞中的表达，并设计针对ALK1的特异性小干扰RNA（siRNA），利用转染试剂将ALK1siRNA瞬时转染至原代培养的健康人皮肤成纤维细胞中。转染后的细胞分两组，一组用无血清培养基OPTI—MEM培养，另一组在OFFI—MEM中加入转化生长因子131（TGFβ1）。培养72h后，Western印迹检测纤连蛋白和PAI-1蛋白量的变化。结果Western印迹和免疫荧光法结果显示，ALK1在健康人和系统性硬化病患者皮肤成纤维细胞中均有表达，且在系统性硬化病患者中的表达量明显高于健康人；免疫荧光结果显示，在成纤维细胞中ALK1位于胞膜和胞质中。转染了ALK1siRNA的成纤维细胞其ALK1、纤连蛋白和PAI-1蛋白表达水平分别下降90％、58％和31％。TGFβ1可显著促进对照siRNA组成纤维细胞表达ALK1、纤连蛋白和PAI-1，而ALK1siRNA则显著抑制TGFβ1诱导的ALK1、纤连蛋白和PAI-1的增加。结论TGF[M可通过ALK1诱导成纤维细胞纤连蛋白和PAI-1的表达，ALK1可能参与了系统性硬化病中纤维化的形成。Objective To measure the expression of activin receptor-like kinases 1 （ALK1） in dermal fibroblasts from patients with systemic scleroderma （SSc） and to estimate its role in the production of fibronectin and plasminogen activator inhibitor-1 （PAI-1）. Methods Dermal fibroblasts were isolated from the lesions of 12 patients with SSe as well as the normal skin of 14 healthy controls, and subjected to a primary culture. The third-passage fibroblasts were used in the next experiment. Western blot and indirect immunofluorescence technique were utilized to quantify the expression of ALK1. A specific siRNA targeting ALK1 was designed, constructed, and transiently transfected into the control dermal fibroblasts, which were then classified into 2 groups to be cultured with or without the presence of transforming growth factor （TGF）-β1 for 72 hours followed by the detection of fibronectin and PAI-1 expression with Western blot. Results As Western blot and direct immunofiuorescence technique showed, both control and SSc fibroblasts showed an expression of ALK1 in the cytoplasm and membrane, and the expression intensity of ALK1 in SSc fibroblasts was significantly higher than that in the control fibroblasts （1.97 ±0.05 vs. 1.12 ± 0.03, t = 50.96, P 〈 0.05）. The expression of ALK1, fibroneetin and PAI-1 was decreased by 90%, 58% and 31% respectively in specific siRNA-transfected SSc fibroblasts compared with the control siRNA-trausfected fibroblasts. TGFβI significantly increased the expression of ALK1, fibronectin and PAI-I in the control siRNA-transfected fihroblasts, but the increase was markedly inhibited by the siRNA-targeting ALK1. Conlusion TGFβI can promote the production of fibroneetin anti PAl1 via ALKI in fibroblasls, and ALKI may be involved in the development of sclerosis in SSe.

关 键 词：人皮肤成纤维细胞 系统性硬化病 激活素 WESTERN印迹 患者 纤溶酶原激活物抑制因子 激酶 受体 

分 类 号：R285.5[医药卫生—中药学]