瘢痕疙瘩成纤维细胞与正常人皮肤成纤维细胞增殖和胶原产生及相关基因的表达

Proliferation, collagen production and related gene expression in keloids and normal skin fibroblasts

目的 探讨人瘢痕疙瘩成纤维细胞与正常人皮肤成纤维细胞增殖、细胞周期、胶原产生及其相关基因表达的差异.方法 选取瘢痕疙瘩患者和正常皮肤美容手术者各12例.分离并原代培养瘢痕疙瘩和正常人皮肤成纤维细胞,对两者细胞动力学、细胞周期分布,p16、p21、p27、Ⅰ型前胶原mRNA表达水平、羟脯氨酸含量、胶原产生进行分析.结果 瘢痕疙瘩成纤维细胞（KFb）外观形态与正常皮肤成纤维细胞（NFb）相似.但羟脯氨酸含量和胶原产生明显高于NFb [（2.30±0.10） μg/ml比（1.66±0.13） μg/ml,（17.19±0.75）μg/ml比（12.37±0.94） μg/ml,均P＜0.05].与NFb相比,KFb处于G2/M期的比例显著增高[（5.90±0.62）％比（16.94％±1.93）％,P＜0.05],而G0/G1期比例则明显低于NFb[（90.24±2.27）％比（75.65±1.92）％,P＜0.05].细胞周期相关基因p16、p21、p27 mRNA表达低于NFb,Ⅰ型前胶原mRNA表达总体高于NFb[0.84±0.11、1.32±0.2、1.69±0.12、4.33±0.27比1.43±0.13、2.56±0.26、2.89±0.37、1.40±0.12,P＜0.05].结论 人瘢痕疙瘩成纤维细胞和正常人皮肤成纤维细胞增殖、细胞周期、胶原产生及其相关基因表达的差异明显.

Objective To study the proliferation,collagen production and related gene expression in keloids and normal skin fibroblast.Methods Isolated primary cells of keloid fibroblasts （KFb,n=12） and normal human dermal fibroblasts （NFb,n=12） were identified,the cell viability and proliferating potential and the cell cycle were detected,and the difference on the collagen synthesis between KFb and NFb were compared.The expression of cell cycle-associated genes such as p21,p16,and p27 was dectected by real-time fluorescent quantitative PCR.Results The phase contrast optical microscopy imaging showed that both KFb isolated from keloid tissues and NFb from normal skin tissues possessed classic and similar fibroblast morphology.But there was a significant difference between cell proliferation,Hyp [（2.30±0.10） μg/ml vs.（1.66±0.13） μg/ml,P〈0.05] and collagen levels [（17.19±0.75） μg/ml vs.（12.37±0.94） μg/ml,P〈0.05].Compared with NFb,KFb exhibited more percentage of G2/M phase cells [（5.90±0.62）% vs.（16.94 %±1.93）%,P〈0.05]and less percentage of G0/G1 phase cells [（90.24 ±2.27）% vs.（75.65±1.92）%,P〈0.05].Cell cycle related genes p16,p21 and p27 were low expressed.Collagen type Ⅰ was highly expressed at mRNA levels in KFb than that in NFb [0.84±0.11,1.32±0.2,1.69±0.12,4.33±0.27 in KFb vs.1.43±0.13,2.56±0.26,2.89±0.37,1.40±0.12 in NFb,P〈0.05].Conclusions There are cell dysfunction and abnormal cellular dynamics in keloid fibroblasts.The formation of keloid likely involves aberrant interactions of some genes that affected its development at different extents.