血管紧张素Ⅱ对转化生长因子β诱导的人皮肤成纤维细胞增殖的影响

目的 观察血管紧张素Ⅱ(angiotensin Ⅱ,Ang Ⅱ)对人皮肤成纤维细胞增殖及对转化生长因子β(transforming growth factor β,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Ang Ⅱ(1×10-10、1×10-9、1×10-8、1×10-7 mol/L)、 TGF-β(0.1、1.0、10.0 ng/ml)、1×10-10 mol/L Ang Ⅱ+ 0.1 ng/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Ang Ⅱ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Ang Ⅱ(1×10-9、1×10-8、1×10-7 mol/L)或TGF-β(1.0、10.0 ng/ml)均能促进成纤维细胞的3H-TdR掺入量(P＜0.05);1×10-10 mol/L Ang Ⅱ或0.1 ng/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).1×10-7 mol/L Ang Ⅱ、10.0 ng/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).1×10-10 mol/L Ang Ⅱ或0.1 ng/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 Ang Ⅱ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.Ang Ⅱ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.

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

OBJECTIVE: To observe the effect of angiotensin II (Ang II) or/and transforming growth factor beta (TGF-beta) 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 x 10(-10), 1 x 10(-9), 1 x 10(-8) and 1 x 10(-7) mol/L) , TGF-beta(0.1, 1.0 and 10.0 ng/ml), and 1 x 10(-10) mol/L Ang II + 0.1 ng/ml TGF-beta, 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 x 10(-9), 1 x 10(-8), 1 x 10(-7) mol/L or TGF-beta at 1.0, 10.0 ng/ml increased 3H-TdR incorporation into cultured skin fibroblasts dose-dependently. Ang II and TGF-beta at lower doses (1 x 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-beta at these doses significantly increased 3H-TdR incorporation into fibroblasts (P<0.05). Ang II at 1 x 10(-7) mol/L or TGF-beta at 10.0 ng/ml significantly increased ERK phosphorylation of fibroblasts after stimulation (P<0.01). Smaller doses of Ang II (1 x 10(-10) mol/L) or TGF-beta (0.1 ng/ml) did not influence ERK phosphorylation of fibroblasts, whereas co-administration of Ang II and TGF-beta 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-beta synergistically increase skin fibroblast proliferation, which is at least partly via enhancement of ERK activity.