血管紧张素对转化生长因子β诱导的人皮肤成纤维细胞增殖的影响

EFFECT OF ANGIOTENSIN Ⅱ ON TRANSFORMING GROWTH FACTOR β-INDUCED FIBROBLAST PROLIFERATION IN HUMAN SKIN

目的观察血管紧张素Ⅱ（angiotensinⅡ，AngⅡ）对人皮肤成纤维细胞增殖及对转化生长因子β（transforming growth factorβ，TGF—β）诱导的成纤维细胞增殖作用的影响，并初步探讨可能的信号机制。方法取自愿捐献的正常皮肤组织标本，采用胶原酶法行成纤维细胞培养。取第4～5代细胞，按实验设计分别加入不同浓度的AngⅡ（1×10^-10、1×10^-9、1×10^-8、1×10^-7mol／L）、TGF-β（0．1、1.0、10．0ng／m1）、1×10^-10mol／L AngⅡ＋0．1ng／ml TGF—β共8组；对照组仅加入等量DMEM。以^3H—TdR掺入法测定细胞增殖，Western blot法检测抗细胞外信号调节激酶（extracellular signal—regulated kinases，ERK）活性变化，观察不同浓度的AngⅡ或／和TGF-β对培养的成纤维细胞^3H—TdR掺入量和ERK磷酸化的影响。结果与对照组比较AngⅡ（1×10^-9、1×10^-8、1×10^-7mol／L）或TGF—β（1．0、10．0ng／ml）均能促进成纤维细胞的^3H—TdR掺入量（P〈0．05）；1×10^-10mol／L AngⅡ或0．1ng／ml TGF—β单独使用不影响成纤维细胞的^3H—TdR掺入量，但二者联合使用提高了成纤维细胞的^3H—TdR掺入量（P〈0．05）。1×10^-7mol／L AngⅡ、10．0ng／ml TGF—β增加皮肤成纤维细胞的ERK磷酸化，与对照组比较差异有统计学意义（P〈0．01）。1×10^-10mol／L AngⅡ或0．1ng／ml TGF-β单独刺激成纤维细胞并未影响ERK磷酸化，而二者联合使用增加ERK磷酸化，与对照组比较差异有统计学意义（P〈0．05）。应用抗ERK抗体显示各组ERK含量一致。结论AngⅡ不仅能作为促有丝分裂素直接促进成纤维细胞分裂增殖，同时也可作为调节因子促进TGF—β的促增殖作用。AngⅡ和TGF—β通过各自特异性受体共同作用于ERK，使磷酸化增加是其产生协同作用可能的机制之一。

Objective To observe the effect of angiotensin Ⅱ （Ang Ⅱ ） or/and transforming growth factor β （TGF-β） on human skin fibroblast proliferation, and to explore the possible signaling mechanism involved in their actions. Methods Cultured human skin fibroblasts were treated with different concentrations of Ang Ⅱ（1 × 10^-10, 1×10^- 9,1×10^- 8and 1×10^-7mol/L） , TGF-β（0. 1, 1.0 and 10.0 ng/ml）, and 1×10^-10mol/L AngⅡ ＋0. 1 ng/ml TGF-β, respectively. The cell proliferation was determined by ^3H-thymidine （^3H-TdR） incorporation. The phosphorylation of extracellular signal-regulated kinases （ERK） was detected by Western blot. Results Ang Ⅱ at 1 × 10^-9, 1 × 10^-8, 1× 10^-7 mol/L or TGF-β at 1.0, 10.0 ng/ml increased ^3H-TdR incorporation into cultured skin fibroblasts dose-dependently. Ang Ⅱ and TGF-β at lower doses （1 × 10^- 10 mol/L and 0.1 ng/ml, respectively） did not affect ^3H-TdR incorporation into fibroblasts （P〉0.05）, whereas co-administration of both Ang Ⅱ and TGF-β at these doses significantly increased ^3H-TdR incorporation into fibroblasts（P〈0.05）. Ang Ⅱ at 1 × 10^-7 mol/L or TGF-β at 10.0 ng/ml significantly increased ERK phosphorylation of fibroblasts after stimulation （P〈0.01）. Smaller doses of Ang Ⅱ （1 × 10^-10 mol/L） or TGF-β （0.1 ng/ml） did not influence ERK phosphorylation of flbroblasts, whereas coadministration of Ang II and TGF-β at these doses significantly enhanced ERK phosphorylation （P〈0. 05）. Total protein levels of ERK did not differ at different doses. Conclusion These results indicate that Ang Ⅱ and TGF-β synergistically increase skin fibroblast proliferation, which is at least partly via enhancement of ERK activity.