【作者】 张俊成；

【导师】 谢有富；

【作者基本信息】 暨南大学 ， 烧伤整形外科， 2010， 硕士

【摘要】 目的：研究增生性瘢痕与体内褪黑素失调和褪黑素受体表达异常的关系,探讨褪黑素对增生性瘢痕成纤维细胞生物活性的影响与机制,为探索防治增生性瘢痕的新方法提供理论依据。方法：1.采用ELISA法测定增生性瘢痕患者血浆、瘢痕水泡液和组织上清液中褪黑素水平。2.免疫组化和荧光定量PCR法检测增生性瘢痕和正常皮肤组织中褪黑素受体的表达。3.利用浓度为10-5mmol/L、10-3mmol/L和1mmol/L褪黑素对增生性瘢痕成纤维细胞进行干预,采用XTT-PMS比色法检测细胞增殖活性,流式细胞仪分析细胞周期和凋亡情况,荧光定量PCR法检测细胞周期素E(cyclin E)、P53和FasmRNA含量的变化。结果：1.增生性瘢痕患者白天血浆褪黑素浓度明显低于健康人白天血浆褪黑素浓度(P<0.05),但患者昼夜血浆褪黑素浓度差异不显著(P>0.05)；瘢痕水泡液中褪黑素浓度明显低于其血浆褪黑素浓度(P<0.05)；瘢痕组织中褪黑素含量显著低于健康皮肤组织中含量(P<0.05)。2.免疫组化显示增生性瘢痕和正常皮肤表皮基底层细胞、毛囊和汗腺均有褪黑素受体GPR50阳性表达,但增生性瘢痕成纤维细胞有广泛阳性表达,而正常皮肤组织中则少见表达。荧光定量PCR显示增生性瘢痕组织中MT1、MT2mRNA含量均高于正常皮肤(P<0.05),同时MT1mRNA含量在两种组织中均高于MT2(P<0.05),基因测序显示扩增产物与GeneBank中人MT1、MT2受体基因序列相吻合。3.体外培养的增生性瘢痕成纤维细胞,随褪黑素浓度升高,细胞集落逐渐散开,细胞体积逐渐缩小,胞膜皱缩,核浆比例减小,核边聚伴有碎裂；褪黑素能抑制瘢痕成纤维细胞增殖,抑制效率随药物浓度升高而增强(P＜0.05)；随褪黑素浓度升高,成纤维细胞G1期比例升高,S和G2期比例则明显降低(P＜0.05),细胞早、晚期凋亡比例均升高(P＜0.05)：成纤维细胞cyclin E mRNA含量随褪黑素浓度升高而降低(P＜0.05),P53和FasmRNA表达则随褪黑素浓度升高而增强(P＜0.05)。结论：1.增生性瘢痕形成与患者体内褪黑素水平下降及组织中褪黑素受体表达升高有关。2.褪黑素能抑制增生性瘢痕成纤维细胞增殖和诱导成纤维细胞凋亡,对增生性瘢痕有一定的防治作用。更多还原

【Abstract】 ObjectiveTo study the relationship between the hypertrophic scarring and melatonin, melatonin receptor. To investigate the effect of melatonin on proliferation and apoptosis of fibroblasts from human hypertrophic scar, as well as to explore the new approach of preventing hypertrophic scar formation.Methods1. The melatonin of the serum, the scar blisters liquid, as well as the tissues lysate supernatant which obtained from hypertrophic scar was quantified by ELISA method.2. The expression of melatonin receptorGPR50 was located with immunohistochemistry and the content of melatonin receptor(MT1、MT2) were assessed with RT-PCR method.The positive production was sequenced with auto sequencing instrument.3. Fibroblasts from hypertrophic scar were cultured and incubated with melatonin at the concentration of 10-5 mmol/L、10-3 mmol/L、1mmol/L for 24h, cell morphology was observed by microscope, and XTT-PMS assay were used to measure the proliferation of fibroblasts. Flow cytometry was used to assess the cell cycle distribution and apoptosis. The content of cell cyclinE、P53 and FasmRNA were determined with RT-PCR method.Results1. The serum melatonin concentrations of hypertrophic scar patients was significantly lower than control group during daytime (P<0.05), there were no significant difference of serum melatonin concentration in patients between the day and night (P>0.05); the melatonin concentration in scar blisters liquid was significantly lower than the serum (P<0.05); and compared with the normal skin tissue, the melatonin in hypertrophic scar was significantly decreased (P<0.05)2. Positive signals of melatonin receptor could be found in the cell membrane and cytoplasm.In normal skin,melatonin receptorGPR50 was located in the epithelial basal cells,sweat gland cells and hair follicle.In human hypertrophic scar,the expression of melatonin receptorGPR50 was mainly existed in the epidermal cells, fibroblasts, survived hair follicle and sweat glands. RT-PCR analysis identified that the expression of melatonin receptor(MT1,MT2)mRNA in hypertrophic scar was significantly stronger than normal skin(P<0.05).In normal skin and hypertrophic scar group, the expression of MT1mRNA was 0.99081±0.26485(copy number/μ1 cDNA) and 1.16584±0.21829(copy number/u1cDNA) respectively; the expression of MT2mRNA was 0.77083±0.15927(copy number/u 1 cDNA) and 0.99550±0.14624(copy number/μu 1 cDNA), respectively. Sequencing results indicated that the positive product coincided with cDNA of human melatonin receptor in GeneBank.3. Compared with the control,melatonin at the concentration of 10-5 mmol/L 10"3 mmol/L、1mmol/L could depress the proliferation of fibroblasts (P<0.05),and the nuclear pyknosis and nuclear fragmentation increased which is concentration-dependent.With the increasing of melatonin concentration, G1 phase cells and the apoptotic rate increased, but S and G2 phase cells decreased (P< 0.05),and the expression of cell cyclin E mRNA were suppressed,but the expression of p53 & FasmRNA were elevated (P<0.05)Conclusions:1. The abnormalities of melatonin and melatonin receptors may be the factors of hypertrophic scarring.2. Melatonin can inhibit the proliferation and induce the apoptosis of hypertrophic scar fibroblasts in vitro, suggesting that melatonin may be effective on preventing hypertrophic scar.更多还原