褪黑素对人增生性瘢痕中成纤维细胞增殖和凋亡的影响

[Effect of melatonin on proliferation and apoptosis of fibroblasts in human hypertrophic scar].

作者信息

Xie You-fu, Zhang Jun-cheng, Liu Si-jun, Dai Li-bing, Du Gao-wei

机构信息

Department of Burns and Plastic Surgery, Ji'nan University Medical School, Guangzhou, China.

出版信息

Zhonghua Shao Shang Za Zhi. 2011 Dec;27(6):422-6.

PMID:22340787

Abstract

OBJECTIVE

To study the effect of melatonin on proliferation and apoptosis of fibroblasts in human hypertrophic scar and its mechanism.

METHODS

Fibroblasts from human hypertrophic scar were isolated and cultured with DMEM medium containing 10% FBS, and then they were divided into control (C, added with ethanol), low concentration (LC, added with 1 × 10(-5) mmol/L melatonin), middle concentration (MC, added with 1 × 10(-3) mmol/L melatonin), and high concentration (HC, added with 1 mmol/L melatonin) groups according to the random number table. After being cultured for 24 hours, cell morphologic change was observed under microscope; XTT-PMS assay was used to examine cell proliferative activity; cell cycle and apoptosis were assessed with flow cytometry after double staining of FITC and PI, and the levels of cyclin E, p53, and Fas mRNA were determined with fluorescence quantitative RT-PCR. Data were processed with analysis of variance and LSD test.

RESULTS

(1) Fibroblasts in C group were spindle-shaped with growth in colonies. Along with the increase in melatonin concentration, fibroblasts in LC, MC, and HC groups gradually dispersed, deformed and atrophied, with shrunk cellular membrane, and decrease in ratio of nucleus and cytoplasm. (2) Proliferative activity of fibroblasts in LC, MC, and HC groups decreased along with an increase in melatonin concentration (1.49 ± 0.15, 1.24 ± 0.20, and 0.92 ± 0.09), which were lower that in C group (1.79 ± 0.10, F = 67.61, P < 0.05). Cell ratios of S and G2/M phases in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were all lower than those in C group [(10.6 ± 1.1)%, (6.1 ± 1.2)%, (3.2 ± 0.8)% vs.(16.9 ± 1.3)%, F = 286.10, P < 0.05; (13.5 ± 1.1)%, (9.8 ± 1.0)%, (6.0 ± 0.7)% vs. (16.7 ± 1.6)%, F = 162.69, P < 0.05]. Apoptotic rates in early and late stages of LC, MC, and HC groups increased along with an increase in melatonin concentration, all higher than those in C group (with F value respectively 424.05, 236.44, P values all below 0.05). The expressions of cyclin E mRNA in LC, MC, and HC groups decreased along with an increase in melatonin concentration, which were lower than that in C group (1.58 ± 0.21, 0.90 ± 0.20, and 0.24 ± 0.12 vs. 2.90 ± 0.30, F = 266.79, P < 0.05), while the expressions of p53 mRNA and Fas mRNA showed opposite tendency (with F value respectively 10.11, 12.03, P values all below 0.05).

CONCLUSIONS

Melatonin can inhibit proliferation and induce apoptosis of fibroblasts in hypertrophic scar through regulating the gene expressions of cyclin E, p53, and Fas.

摘要

目的

研究褪黑素对人增生性瘢痕成纤维细胞增殖及凋亡的影响及其机制。

方法

分离人增生性瘢痕成纤维细胞，用含10%胎牛血清的DMEM培养基培养，然后按随机数字表法分为对照组（C组，加入乙醇）、低浓度组（LC组，加入1×10⁻⁵ mmol/L褪黑素）、中浓度组（MC组，加入1×10⁻³ mmol/L褪黑素）和高浓度组（HC组，加入1 mmol/L褪黑素）。培养24小时后，在显微镜下观察细胞形态变化；采用XTT-PMS法检测细胞增殖活性；经FITC和PI双染后用流式细胞术检测细胞周期及凋亡情况，并用荧光定量RT-PCR检测细胞周期蛋白E、p53和Fas mRNA的水平。数据采用方差分析和LSD检验进行处理。

结果

（1）C组成纤维细胞呈梭形，集落生长。随着褪黑素浓度增加，LC组、MC组和HC组成纤维细胞逐渐分散、变形、萎缩，细胞膜皱缩，核质比减小。（2）LC组、MC组和HC组成纤维细胞的增殖活性随褪黑素浓度增加而降低（分别为1.49±0.15、1.24±0.20、0.92±0.09），均低于C组（1.79±0.10，F = 67.61，P < 0.05）。LC组、MC组和HC组S期和G2/M期细胞比例随褪黑素浓度增加而降低，均低于C组[（10.6±1.1）%、（6.1±1.2）%、（3.2±0.8）%对（16.9±'1.3）%，F = 286.10，P < 0.05；（13.5±1.1）%、（9.8±1.0）%、（6.0±0.7）%对（16.7±1.6）%，F = 162.69，P < 0.05]。LC组、MC组和HC组早期及晚期凋亡率随褪黑素浓度增加而升高，均高于C组（F值分别为424.05、236.44，P值均小于0.05）。LC组、MC组和HC组细胞周期蛋白E mRNA表达随褪黑素浓度增加而降低，低于C组（1.58±0.21、0.90±0.20、0.24±0.12对2.90±0.30，F = 266.79，P < 0.05）'，而p53 mRNA和Fas mRNA表达呈相反趋势（F值分别为10.11、12.03，P值均小于0.05）。