AMPK激动剂干预瘢痕疙瘩形成过程的实验研究

目的探索AMPK激动剂对瘢痕疙瘩(Keloid)形成过程的干扰作用,为瘢痕疙瘩的临床治疗提供新的思路。方法通过缺氧环境及不同浓度(0 ng/m L、2.5 ng/m L、5 ng/m L、10 ng/m L和20.0 ng/m L)TGF-β1诱导人皮肤成纤维细胞表型向瘢痕疙瘩相关细胞表型转化,并通过细胞形态学及Western blot检测来验证诱导结果。随后加入不同剂量(2.5μmol、5μmol、10μmol和20μmol)AMPK激动剂(A769662),通过细胞形态学观察、Western blot检测及ELISA检测,观察AMPK激动剂对人皮肤成纤维细胞表型向瘢痕疙瘩相关细胞表型转化的干预作用。结果在缺氧条件下及加入不同浓度TGF-β1后,人皮肤成纤维细胞形态会发生改变,纺锤形态丧失;在TGF-β1浓度不断增加至10 ng/m L的过程中,TGF-β1刺激人皮肤成纤维细胞增殖并向肌成纤维细胞转化;而浓度继续增加时,效果出现停滞并有逆转趋势;在同一TGF-β1浓度诱导下,随着加入AMPK激动剂剂量的增加,人皮肤成纤维细胞表型向瘢痕疙瘩相关细胞表型转化过程被逐步干预;在缺氧环境下,人皮肤成纤维细胞中VEGF和IL-6的表达均明显提高,在TGF-β1诱导下,VEGF和IL-6的合成进一步增加,而随着AMPK激动剂的添加,VEGF和IL-6的表达逐步减少。结论 AMPK激动剂能干预由缺氧及TGF-β1诱导的人皮肤成纤维细胞表型向瘢痕疙瘩相关细胞表型转化的过程。

The Inhibition Effect of AMPK Agonist on the Formation of Keloid

Objective To explore the interference effect of AMPK agonist on the formation of keloid, and to provide new ideas for the clinical treatment of keloid. Methods In vitro culture conditions of skin fibroblasts phenotype to the keloid phenotype transformation were established by hypoxia and different concentrations （0, 2.5, 5, 10, 20 ng/mL） of TGF-β1, and confirmed by cell morphology and Western blot. Subsequently, different doses （2.5, 5, 10, 20 μmol） of AMPK agonist （A769662） were added to observe its effect on the phenotypic transformation of skin fibroblast to keloid. The reserve effect was confirmed by cell morphology, the expression of related markers （E-cadherin, Vimentin, ACTA2, Collagen I , IRS-1, Glut4, p-H2AX/H2AX2, NANOG, OCT4） detected by Western blot and the expression of IL-6 and VEGF detected by ELISA. Results The spindle shape of skin fibroblasts lost under hypoxia and inflammation induction by TGF-β1. In the process of increasing TGF-β1 concentration to 10 ng/mL, TGF-β1 stimulated the transformation of phenotype of fibroblasts into keloid associated phenotype. As the concentration continued to increase, the effects were arrested and slightly reversed. At the same concentration （10 ng/mL） induced by TGF-β1, the process of cell transformation was gradually intervened with the increasing dose of AMPK agonists added. Under hypoxia condition, the expression of VEGF and IL-6 in skin fibroblasts was significantly increased. Under the induction of TGF-β1, the expression of VEGF and IL-6 was further increased. And with the addition of AMPK agonists, the expression of VEGF and IL-6 was progressively reduced. Conclusion AMPK agonists can inhibit the transformation of fibroblast to keloid associated phenotype induced by hypoxia and inflammation.