病理性瘢痕成纤维细胞的增殖调控及Fas基因突变的筛选

目的 从细胞生物学及基因水平探讨正常皮肤、增生性瘢痕、瘢痕疙瘩周边部和中央部不同生长特性及瘢痕疙瘩发生的可能机制。方法 （１）取手术切除的正常皮肤、增生性瘢痕和瘢痕疙瘩组织各６例为标本,通过细胞培养６～１０代后,应用流式细胞仪、粘附式细胞仪检测不同来源的成纤维细胞ｐ５３蛋白的表达和细胞周期的分布。（２）取瘢痕疙瘩、增生性瘢痕患者的组织及外周血标本,聚合酶链式反应（ＰＣＲ）特异性扩增Ｆａｓ分子外显子

The distrubution of cell cycle on fibrolasts derived from the pathlogical scars and analysis of Fas gene mutations in keloids using polymerse chain Reaction based single strand conformation polymorphism

OBJECTIVE: To explore the concrete mechanism which accounts for the different growth characteristics of normal skins and pathlogical scars and to identify if the mutations of Fas gene exist. METHODS: Six samples of normal skin, hypertrophic scars and keloids were collected. The means of cell culture was used, and only 6 - 10 passages fibroblasts were selected for experiment. The distribution of cell cycle and expression of protein p53 were analysed with flow cytometry, adherant cell analysis and sorting interactive laser coytometry (ACAS 570). Keloids and hypertrophic scar tissue and the peripheral blood of each patient were collected. After DNA was isolated from the samples, we used the single-stranded conformation polymorphism (SSCP) analysis and DNA sequencing to examine the structure of Fas gene. RESULTS: The expression of p53 protein on the fibroblasts derived from normal skin was significantly elevated in comparison to the hypertrophic scars with lowest propagative fibroblasts. A large percent of fibroblasts derived from the peripherial areas of keloids distributed in G(2), S and M periods with low levels of p53 protein. On the other hand, fibroblasts derived from the central areas of keloids distributed mostly in G(0) and G(1) periods with higher expression of p53 protein. Mutation in Fas gene was identified and confirmed in keloid tissue of 20% (2/10) of keloid patient, which may cause loss of function and contribute to the pathogenesis of keloid. A deletion of the nucleotide "A" at the same position was found, which caused a frameshift mutation in exon 9. CONCLUSION: The difference of the distributions of cell cycle and expression of protein p53 may account for the different growth characteristics in pathlogical scar and normal skin. Mutations in Fas gene may lead to abnormal apoptosis of fibroblasts in keloids, and may contribute to the pathogenesis and progression of keloids.