Fas介导瘢痕疙瘩周围皮肤成纤维细胞凋亡及其基因检测研究

目的　探讨瘢痕疙瘩周围皮肤中是否有生物学活性异常成纤维细胞 ,以期进一步了解瘢痕疙瘩的发展机制。方法　所取新鲜组织标本进行细胞培养 ,通过碘化丙啶 (PI)染色、激光流式细胞仪比较不同浓度Fas单克隆抗体 (FasMcAb ,0～ 10 0 0 μg/L)作用后各组成纤维细胞凋亡率。采用聚合酶链反应 (PCR)技术对Fas基因外显子 8进行DNA扩增并测序。结果　FasMcAb作用2 4h后 ,瘢痕疙瘩成纤维细胞在各浓度段均不能凋亡 ,瘢痕疙瘩周围皮肤成纤维细胞随FasMcAb作用浓度的增加凋亡率虽有所增加 ,但总体比较与瘢痕疙瘩差异无显著性 (P >0 .0 5 ) ,而与正常皮肤差异有显著性 (P <0 .0 1)。瘢痕疙瘩周围皮肤 6例标本中有 4例 (4 /6)Fas基因外显子 8及其下游序列存在点突变及移码突变 ,均与同患者瘢痕疙瘩细胞基因序列分析结果一致 ,而两组正常皮肤成纤维细胞则未发现有任何形式的基因突变。结论　瘢痕疙瘩周围 0 .5cm皮肤内存在生物学活性异常细胞 ,这可能为瘢痕疙瘩浸润性生长的结果及其治疗后易复发的原因之一。

Apoptosis induced by FasMcAb and detection of gene mutations of Fas exon 8 of fibroblasts derived from the surrounding skin of keloids

Objective To investigate if there are abnormal fibroblasts derived from the skin surrounding keloids in order to better understand the keloids' progression.Methods All of samples were used for cell culture.Flow cytometry were used for comparing the apoptotic rate of fibroblasts derived from keloids and its surrounding skin when it was induced by Fas antibody.Fas exon 8 was amplified by PCR and DNA sequenced to examine the structure of gene of destination.Results When induced by Fas antibody,the apoptotic rate of fibroblasts derived from the surrounding skin of keloids was increased as compared with that in normal skin (P<0.01).In general,there were no significant differences in cell apoptotic rate between keloids and its surrounding skin (P>0.05).Mutations (point and frameshift mutations) of Fas exon 8 (4/6) in fibroblasts derived from the surrounding skin of keloids were identified.Fibroblasts derived from keloids had the same mutations as its surrounding skin.Conclusion There are abnormal fibroblasts in the surrounding skin of keloids.Maybe it is the result of keloids' infiltrative growth and one of the reasons for easy recurrence after therapy.