新型退化电波形刺激联合光动力疗法可显著增强瘢痕成纤维细胞的细胞毒性作用：一种潜在联合治疗方法的首次报道。

Addition of novel degenerate electrical waveform stimulation with photodynamic therapy significantly enhances its cytotoxic effect in keloid fibroblasts: first report of a potential combination therapy.

机构信息

Plastic & Reconstructive Surgery Research, School of Translational Medicine, Manchester Interdisciplinary Biocentre (MIB), The University of Manchester, UK.

出版信息

J Dermatol Sci. 2011 Dec;64(3):174-84. doi: 10.1016/j.jdermsci.2011.08.012. Epub 2011 Sep 17.

DOI:10.1016/j.jdermsci.2011.08.012

PMID:22015050

Abstract

BACKGROUND

We recently reported use of photodynamic therapy (PDT) for treating keloid disease (KD). However, in view of high recurrence rates post any treatment modality, adjuvant therapies should be considered. Additionally, we previously demonstrated the effect of a novel electrical waveform, the degenerate wave (DW) on differential gene expression in keloid fibroblasts.

OBJECTIVE

In this study, we evaluated the in vitro cytotoxic effect of PDT at 5J/cm(2) and 10J/cm(2) of red light (633 ± 3nm) using 5-aminolevulinic acid (ALA) and methyl aminolevulinate (MAL) with and without DW, on keloid fibroblasts compared to normal skin fibroblasts.

METHODS

The rate of intracellular photosensitizer (protoporphyrin IX, PPIX) generation and disintegration, reactive oxygen species (ROS) generation, LDH cytotoxicity, WST-1 cytoproliferation, apoptosis by Caspase-3 activation, mitochondrial membrane potential assessment by JC-1 aggregates, qRT-PCR, flow cytometry and In-Cell Western Blotting were performed.

RESULTS

PPIX accumulation and disintegration rate was higher in keloid than normal fibroblasts after incubation with MAL compared to ALA. Increased cytotoxicity and decreased cytoproliferation were observed for keloid fibroblasts after PDT+DW treatment compared to PDT alone. ROS generation, mitochondrial membrane depolarization, apoptosis (Caspase-3 activation) and collagens I and III gene down-regulation were higher in keloid compared to normal skin fibroblasts after MAL-PDT+DW treatment. An increase in the number of cells entering apoptosis and necrosis was observed after PDT+DW treatment by flow cytometry analysis. All positive findings were statistically significant (P<0.05).

CONCLUSION

The cytotoxic effect of PDT on keloid fibroblasts can be enhanced significantly with addition of DW stimulation, indicating for the first time the utility of this potential combinational therapy.

摘要

背景

我们最近报道了光动力疗法（PDT）治疗瘢痕疙瘩（KD）的应用。然而，鉴于任何治疗方法后都有很高的复发率，应考虑辅助治疗。此外，我们之前证明了新型电波形退化波（DW）对瘢痕疙瘩成纤维细胞差异基因表达的影响。

目的

在这项研究中，我们评估了在红色光（633±3nm）5J/cm2和 10J/cm2 时，单独使用 PDT 以及 PDT 联合 DW 对 5-氨基酮戊酸（ALA）和甲氨基酮戊酸（MAL）作用下的瘢痕疙瘩成纤维细胞和正常皮肤成纤维细胞的体外细胞毒性作用。

方法

通过测定原卟啉 IX（PPIX）的生成和分解、活性氧（ROS）生成、乳酸脱氢酶（LDH）细胞毒性、WST-1 细胞增殖、Caspase-3 激活的细胞凋亡、线粒体膜电位（通过 JC-1 聚集体评估）、qRT-PCR、流式细胞术和细胞内 Western 印迹来评估细胞内光敏剂（PPIX）的生成和分解率、ROS 生成、LDH 细胞毒性、WST-1 细胞增殖、Caspase-3 激活的细胞凋亡、线粒体膜电位（通过 JC-1 聚集体评估）、qRT-PCR、流式细胞术和细胞内 Western 印迹。

结果

与 ALA 相比，MAL 孵育后瘢痕疙瘩成纤维细胞的 PPIX 积累和分解率更高。与单独 PDT 相比，PDT+DW 治疗后瘢痕疙瘩成纤维细胞的细胞毒性增加，细胞增殖减少。与正常皮肤成纤维细胞相比，MAL-PDT+DW 治疗后，瘢痕疙瘩成纤维细胞的 ROS 生成、线粒体膜去极化、细胞凋亡（Caspase-3 激活）以及胶原 I 和 III 基因下调更高。通过流式细胞术分析，观察到 PDT+DW 治疗后细胞进入凋亡和坏死的数量增加。所有阳性发现均具有统计学意义（P<0.05）。