瘢痕疙瘩皮损及其周边皮肤干细胞的分布和增殖的比较

Distribution and Proliferation of Stem Cells in Keloid Lesion and its Surrounding Skin

目的观察瘢痕疙瘩皮损处、边缘处及其周边外观正常组织中来源细胞的类干细胞特性。方法分别从瘢痕疙瘩皮损处、边缘处及手术切缘外0.5 cm的外观正常组织中分离真皮细胞,进行原代培养。采用实时定量PCR(Quantitative Real-time PCR,qRT-PCR)及流式细胞术检测部分间充质干细胞、造血干细胞及胚胎干细胞相关表型标志物。成球实验根据球体数量来评估自我增殖及形成细胞克隆能力。CCK-8法检测上述三处原代细胞对丝裂霉素C耐药性差异。结果流式细胞术显示瘢痕疙瘩皮损处、边缘处及切缘外外观正常组织来源的原代细胞均高表达间充质干细胞表型标志物(CD13、CD44、CD90),低表达造血干细胞表型标志物CD34。qRT-PCR显示C-kit在瘢痕疙瘩中的表达低于正常组织,其余造血干细胞、间充质干细胞、胚胎干细胞等表型标志物在三处来源细胞之间均未见明显差异。此外,对铺板的6000个细胞形成的球体数量进行分别计数,瘢痕疙瘩皮损处为(22.13±5.62)个,边缘处为(16.73±4.6)个,周边外观正常组织来源为(20.42±2.01)个,三处间差异无统计学意义(P>0.05)。丝裂霉素C(0.0025~0.08 mg/L)对三处来源细胞均有显著的抑制作用,在低浓度(0.05 mg/L)时,瘢痕疙瘩来源细胞较周边外观正常组织来源细胞具有更显著的耐药性(P<0.05)。结论人瘢痕疙瘩皮损处、边缘处及周边正常组织来源细胞均显著表达间充质干细胞表型。

Objective The current study aimed to observe stem-like characteristics of cells from keloid lesions,margins and the surrounding normal tissues.Methods Cells were isolated and primary cultured from keloid lesions,margins and the normal tissue,0.5 cm outside the surgical margins.The phenotypic markers of mesenchymal stem cells(MSC),hematopoietic stem cells(HSC)and embryonic stem cells(ESC)were analyzed by real-time quantitative PCR(qRT-PCR)and flow cytometry.The self-proliferation ability and cell cloning formation was evaluated using sphere formation assay according to the number of spheres.CCK-8 assay was employed to detect the resistance of cells in different groups to mitomycin C.Results The cells from keloid lesions,margins and the surrounding normal tissues present a high expression of the phenotypic markers CD13,CD44 and CD90,which is usually found in MSC.Meanwhile,we found a low expression of CD34,which is usually observed in ESC.The expression of C-kit in keloid was lower than that in normal tissue.In addition,there are no significant differences of cell related phenotypic markers between the cells from MSC,HSC,and ESC among the three source cells.Furthermore,in the sphere culture,there are 22.13±5.62 in keloid lesions,16.73±4.6 in margins,and 20.42±2.01 in surrounding normal tissues.There is no statistical significance between the three tissues(P>0.05).Mitomycin C had a significant inhibitory effect on cells isolated from all three tissues.Low mitomycin C concentration could induce more significant drug resistance in Keloid derived cells compared with the cells isolated from surrounding normal tissues(P<0.05).Conclusion The cells derived from keloid lesions,margins and the surrounding normal tissues expressed immunopheno type of MSC significantly.