褪黑素对人增生性瘢痕成纤维细胞结缔组织生长因子表达的影响  

Effect of melatonin on the expression of connective tissue growth factor of fibroblasts from human hypertrophic scar

作　　者：戴丽冰[1] 谢有富[2] 姚依村 刘思隽[2] 徐家科 刘志河[1] 沈雁[1] 

机构地区：[1]暨南大学医学院第四附属医院广州市红十字会医院广州市创伤外科研究所,广州510220 [2]暨南大学医学院第四附属医院广州市红十字会医院烧伤科,广州510220 [3]暨南大学医学院第四附属医院广州市红十字会医院骨科,广州510220 [4]西澳大利亚大学外科学院病理学和医学实验室

出　　处：《中华实验外科杂志》2015年第3期486-489,共4页Chinese Journal of Experimental Surgery

基　　金：国家自然科学基金资助项目（81272222、81228013）;广东省自然科学基金资助项目（S2013010015184）;广东省科技厅一般引导项目（20128031800338、20138021800042、20138021800070）;广州市科技计划项目（2013J4100099、2013J4100100、2014J4100075）;广州市医药科技一般引导项目（20121A011046、20131A010007）

摘　　要：目的 观察褪黑素对人增生性瘢痕成纤维细胞结缔组织生长因子（CTGF）表达的影响.方法 组织块法分离培养原代人增生性瘢痕成纤维细胞（HSFB）,取第4代细胞分成4组：对照组、褪黑素（Melatonin） 10^-3mmol/L组、褪黑素受体拮抗剂（Luzindole）1mmol/L组、Luzindole1 mmol/L＋ Melatonin 10^-3 mmol/L组,分组培养48 h,采用实时荧光定量聚合酶链反应（FQ-PCR,SYBR Green）检测细胞裂解液CTGF、α-平滑肌肌动蛋白（α-SMA） mRNA表达;细胞爬片免疫荧光细胞化学方法分析CTGF、Ⅰ型胶原（COL-Ⅰ）含量;酶联免疫吸咐试验（ELISA）法检测细胞培养液CTGF、COL-Ⅰ、Ⅲ型胶原（COL-Ⅲ）含量.结果 10-3 mmol/L褪黑素降低增生性瘢痕成纤维细胞CTGF、α-SMA mRNA表达及CTGF、COL-Ⅰ的含量（P＜0.05）,但COL-Ⅲ没影响.对照组、褪黑素组、Luzindole组、Luzindole＋褪黑素组CTGF和α-SMA mRNA分别为0.1294±0.006 1、0.086 9±0.003 1、0.1235±0.008 1、0.123 2±0.0094（F=30.130,P ＜0.01）和0.1347 ±0.0042、0.0847±0.003 1、0.128 9±0.002 6、0.128 2±0.004 1 （F=168.500,P＜0.01）;CTGF和COL-Ⅰ荧光细胞化学相对表达量为0.0135±0.0003、0.0084±0.0003、0.0126±0.0005、0.012 5±0.0004 （F=140.750,P ＜0.01）和0.0127±0.0008、0.0086±0.0004、0.0120±0.0009、0.0120±0.000 8（F=24.240,P＜0.01）;CTGF和COL-Ⅰ细胞培养液浓度（ng/L）为30.0569±1.2869、19.4254±0.924 5、26.673 8±0.9405、25.398 0±0.8478 （F=76.490,P＜ 0.01）和30.0445±1.1575、21.798 5±1.1260、26.5168±1.2923、25.459 4±1.705 7 （F=25.650,P＜ 0.01）;COL-Ⅲ细胞培养液浓度（ng/L）为47.141 4±3.532 6、53.498 2±4.589 9、52.532 5±5.239 8、46.427 1±4.204 2（F=2.680,P＜0.01）.结论 褪黑素通过与受体结合,下调增生性瘢痕成纤维细胞CTGF、α-SMA mRNA表达,从而降低CTGF、COL-Ⅰ的含量.Objective To investigate the effect of melatonin on the expression of connective tissue growth factor （CTGF） of fibroblasts from human hypertrophic scar.Methods Cultured primary fibroblasts cells by using enzyme digestion technique.Fibroblasts were cuhured and incubated with melatonin,Luzindole,melatonin and Luzindole for 48 hours.Fluorescent quantitative polymerase chain reaction assay （FQ-PCR） were used to detect the gene expression of CTGF and α-smooth muscle actin （α-SMA）.Immunofluorescence cytochemistry were used to quantify the expression of CTGF and collagen-Ⅰ （COL-Ⅰ）.In order to study the extracellular matrix expression of CTGF,COL-Ⅰ and COL-Ⅲ,enzyme linked immunosorbent assay （ELISA） was performed.Results The expression of CTGF mRNA in control group,melatonin group,luzindole group and luzindole ＋ melatonin group was 0.129 4 ±0.006 1,0.086 9 ±0.003 1,0.123 5 ±0.008 1,0.123 2 ±0.009 4 （F=30.130,P〈0.01）,while the α-SMA was 0.134 7 ±0.004 2,0.084 7 ±0.003 1,0.128 9 ±0.002 6,0.128 2 ±0.004 1 （F=168.500,P〈 0.01） ; The optical density of CTGF in control group,melatonin group,luzindole group and luzindole ＋ melatonin group was 0.013 5 ± 0.000 3,0.008 4 ± 0.000 3,0.012 6 ± 0.000 5,0.012 5 ± 0.000 4 （F=140.750,P〈0.01）,and the COL-Ⅰ was 0.0127±0.0008,0.0086±0.0004,0.0120± 0.000 9,0.012 0 ± 0.000 8 （F =24.240,P 〈 0.01）.The expression of CTGF （ng/L） in extracellular matrix in control group,melatonin group,luzindole group and luzindole ＋ melatonin group was 30.056 9 ± 1.286 9,19.425 4±0.924 5,26.673 8 ±0.940 5,25.398 0±0.847 8 （F=76.490,P〈0.01）,and the COL-Ⅰ （ng/L） was 30.044 5 ± 1.157 5,21.798 5 ± 1.126 0,26.516 8 ± 1.292 3,25.459 4 ± 1.705 7 （F =25.650,P 〈 0.01）.The expression of COL-Ⅲ （ng/L） in extracellular matrix in these groups was 47.141 4 ± 3.532 6,53.498 2 ± 4.589 9,52.532 5 ± 5.239 8,46.427 1 ± 4.204 2 （F =2.680,P 〉 0.05）.Compared with control group,melatonin decreased the gene express

关 键 词：褪黑素 人增生性瘢痕成纤维细胞 结缔组织生长因子 

分 类 号：R622[医药卫生—整形外科]