人瘢痕疙瘩来源干细胞的生物学特性鉴定

Identification of biological characteristics of human keloid-derived stem cells

目的分析人瘢痕疙瘩来源干细胞的生物学特性。为进一步研究该细胞在瘢痕疙瘩形成中的作用提供参考。方法以人瘢痕疙瘩为研究对象，采用酶消化法及传代培养法分离筛选瘢痕疙瘩于细胞。选取原代和（或）第3代贴壁细胞进行生物学特性鉴定：加入CD29-藻红蛋白（PE）、CD34－PE、CD44-异硫氰酸荧光素（FITC）、CD90-FITC、CD45-多甲藻黄素叶绿素蛋白抗体，流式细胞仪检测细胞表面分子标记物CD29、CD34、CD44、CD45及CD90的表达及细胞周期；加入小鼠抗人细胞角蛋白19（CK19）即用型单克降抗体、鼠抗波形蛋白即用型单克隆抗体，免疫细胞化学法检测CK19、波形蛋白的表达；RT—PCR检测细胞Oct4的表达。取第1代细胞，应用成骨细胞、成脂肪细胞、软骨细胞诱导分化培养液进行诱导分化实验，观察细胞的多向分化能力。结果传代培养后，细胞形态较为均一，以梭肜为主，排列不规则。流式细胞仪检测表明，该细胞高表达CD29、CD44、CD90等间充质干细胞表面标记物，低表达CD34、CD45等造血干细胞表面标记物。细胞周期分析显示67．66％的细胞处于G0／G1期，26．24％的细胞处于G2／M期，6．11％的细胞处于s期。免疫细胞化学法检测显示细胞波形蛋白表达呈阳性、CK19表达呈阴性。RT-PCR法检测显示细胞Oct4表达呈阳性。诱导分化实验表明，细胞可向成骨细胞、软骨细胞和成脂肪细胞分化，具有多向分化潜能。结论人瘢痕疙瘩内存在间充质样于细胞，这种干细胞可能在瘢痕疙瘩形成中发挥重要作用。

Objective To investigate the hiological characteristics of human keloid-derived stem cells （KDSC） in order to further research its role in keloid pathogenesis. Methods Human keloid specimens were harvested to isolate and select KDSC by enzyme digestion and subcuhnring. Primary and （or） the third generation of KDSC were collected for identification of biological characteristics as follows. （ 1 ） After addition of mouse anti-human nmnoclonal fluorescent antibodies （ CD29-PE, CD34-PE, CD44-FITC, CD90- FITC, CD45-PerCP）, the expression of cell surface antigen phenotype （CD29, CD34, CD44, CD45, CD90） as well as cell cycle was analyzed by flow eytometry. （2） After addition of mouse anti-human cell keratin （CK19） monoclonal antihody and mouse anti-human vimentin monoclonal antibody, the expression level of CK19 and vimentin was respectively determined with immunoeytoehemical method. RT-PCR was used to detect the expression of Oct4. The multipotent differentiation capacity of the first generation KDSC was observed with osteogenic, chondrogenic and adipogenic nutrient media. Results Alter being subcultured, the sizes of cells were similar, and the majority of them were spindle-shaped with disorderly arrangement. The cells highly expressed typical surface markers of mesenchymal stem cells （such as CD29, CD44, and CD90, etc. ） with low expression of hematopoietic stem cell surface markers （ such as CD34, CD45, etc. ）. 67.66% of cells were in G0/G1 phase, 26.24% of cells were in G2/M phase, and 6.11% of cells were in S phase. Vimentin was positively expressed in KDSC while CK19 was negatively expressed. The expression of Oct4 was also positive. After being cultured in inducing differentiation media, the cells could differentiate into osteohlasts, chondrocytes, and adipocytes. Conclusions Stem cells existing in human keloid, which are similar to mesenchymal stem cells, may play an important role in keloid pathogenesis.