ABSTRACT

Capacitive-resistive electric transfer (CRET) therapies have been proposed as strategies for regeneration of cutaneous tissue lesions. Previous studies by our group have shown that intermittent stimulation with 448 kHz CRET currents at subthermal densities promotes in vitro proliferation of human stem cells involved in tissue regeneration. The present study investigates the effects of the in vitro exposure to these radiofrequency (RF) currents on the proliferation and migration of keratinocytes and fibroblasts, the main cell types involved in skin regeneration. The effects of the electric stimulation on cell proliferation and migration were studied through XTT and wound closure assays, respectively. The CRET effects on the expression and location of proteins involved in proliferation and migration were assessed by immunoblot and immunofluorescence. The obtained results reveal that electrostimulation promotes proliferation and/or migration in keratinocytes and fibroblasts. These effects would be mediated by changes observed in the expression and location of intercellular adhesion proteins such as β-catenin and E-cadherin, of proteins involved in cell-to-substrate adhesion such as vinculin, p-FAK and the metalloproteinase MMP-9, and of other proteins that control both processes: MAP kinases p-p38, p-JUNK and p-ERK1/2. These responses could represent a mechanism underlying the promotion of normotrophic wound regeneration induced by CRET. Indeed, electric stimulation would favor completion of granulation tissue formation prior to the closure of the outer tissue layers, thus preventing abnormal wound cicatrization or chronification.

摘要

电容电阻电转移 (CRET) 疗法已被提议作为皮肤组织病变再生的策略。我们小组以前的研究表明，在亚热密度下用 448 kHz CRET 电流间歇性刺激可促进参与组织再生的人类干细胞的体外增殖。本研究调查了体外暴露于这些射频 (RF) 电流对角质形成细胞和成纤维细胞增殖和迁移的影响，角质形成细胞和成纤维细胞是参与皮肤再生的主要细胞类型。分别通过 XTT 和伤口闭合试验研究了电刺激对细胞增殖和迁移的影响。通过免疫印迹和免疫荧光评估 CRET 对参与增殖和迁移的蛋白质的表达和定位的影响。获得的结果表明电刺激促进角质形成细胞和成纤维细胞的增殖和/或迁移。这些影响将通过观察到的细胞间粘附蛋白（如 β-连环蛋白和 E-钙粘蛋白）的表达和位置变化、参与细胞与底物粘附的蛋白质（如纽蛋白、p -FAK 和金属蛋白酶 MMP-9，以及控制这两个过程的其他蛋白质：MAP 激酶p -p38、p -JUNK 和p -ERK1/2。这些反应可能代表了促进 CRET 诱导的正常营养伤口再生的潜在机制。事实上，电刺激将有利于在外组织层闭合之前完成肉芽组织的形成，从而防止异常的伤口瘢痕形成或慢性化。