褪黑激素对人增生性瘢痕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

目的探讨褪黑激素对人增生性瘢痕Fb增殖和凋亡的影响与机制。方法采用组织块法分离培养人增生性瘢痕Fb。按随机数字表法将细胞分为低、中、高浓度组和对照组。前3组细胞分别采用含1×10^-5、1×10^-3、1mmol／L褪黑激素的培养液培养，对照组不加褪黑激素常规培养。处理后24h进行如下检测：对各组细胞进行形态学观察；用四氮唑复合物（XTT）-硫酸酚嗪甲酯（PMS）比色法检测细胞增殖活性；对细胞行膜联蛋白V-异硫氰酸荧光素（FITC）和碘化丙啶（PI）双染色后，用流式细胞仪分析细胞周期和凋亡情况；荧光定量RT—PCR法检测细胞周期蛋白E（cyclinE）、p53和FasmRNA表达量。对数据行方差分析和LSD检验。结果形态学观察显示，对照组Fh为长梭形，呈集落分布；3个浓度组Fb随着褪黑激素浓度升高，细胞逐渐分散，胞体变形缩小，胞膜皱缩，核质比例减小。对照组、低浓度组、中浓度组、高浓度组Fb增殖活性（吸光度值）依次下降，分别为1．79．±0．10、1．49±0．15、1．24±0．20、0．92±0．09（F=67．61，P〈0．05）；S期细胞百分比依次下降，分别为（16．9±1．3）％、（10．6±1．1）％、（6．1±1．2）％、（3．2±0．8）％（F=286．10，P〈0．05）；G2／M期细胞百分比依次下降，分别为（16．7±1．6）％、（13．5±1．1）％、（9．8±1．0）％、（6．0±0．7）％（F：162．69，P〈0．05）；早、晚期凋亡细胞百分比依次升高（F值分别为424．05、236．44，P值均小于0．05）。对照组、低浓度组、中浓度组、高浓度组细胞cyclinE的mRNA表达量依次下降，分别为2．90±0．30、1．58±0．21、0．90±0．20、0．24±0．12（F=266．79，P〈0．05）；p53和FasmRNA表达量依次升高（F值分别为10．11、12．03，P值均小于0．05）。结论褪黑激素可通过影响细胞cyclinE、p53和Fas基因的表达，抑制增生性瘢痕Fb增殖并诱导该细胞凋亡。

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 spin- dle-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 de- crease 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 orS 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.