摘要
目的探讨人增生性瘢痕来源的成纤维细胞的细胞表型及分化潜能，以进一步研究其在增生性瘢痕形成中的作用。方法从手术切除的增生性瘢痕中提取并分离培养成纤维细胞，以倒置显微镜观察其细胞形态及生长特点。待细胞培养传代至第3代，采用Vi—CELL细胞计数仪检测细胞生长情况，流式细胞仪检测成纤维细胞间充质干细胞表型标志CD73、CD44、CD105、CD90、CD34、CD45、CDl4的表达情况；通过免疫细胞化学检测该细胞CKl9、Oct4、Vementin的表达情况及免疫荧光检测a-平滑肌肌动蛋白；诱导分化检测细胞向成骨、成软骨和成脂细胞方向分化能力。结果细胞原代培养初期贴壁散在分布，生长曲线显示细胞1～2d生长较慢，从3～5d左右细胞生长较快，6～7d细胞进入平台期；第2代第5天细胞多为长梭形，呈放射状、漩涡状排列。细胞流式结果显示细胞表型CD90、CD44、CDl05、CD73呈高表达，CD45、CD34、CDl4不表达；免疫细胞化学检测间充质细胞标记物Vementin、多能干细胞标志物Oct4均呈阳性表达，免疫荧光检测OL一平滑肌肌动蛋白呈阳性表达，上皮细胞标志物CKl9呈阴性表达，多向诱导分化实验该细胞可向成骨、成软骨和成脂细胞分化，具有多向分化潜能。结论人增生性瘢痕来源成纤细胞具有间充质干细胞样生物学特性，该生物学特性可能在增生性瘢痕的形成以及创面修复中发挥至关重要的作用。
Objective To explore the CD phenotypic, protein expression and pluripotent differentiation of human hypertrophic scar fibroblasts cultured in vitro, so as to study the mechanisms of scar formation. Methods Fibroblasts were isolated and cultured from human hypertrophic scar of 3 cases. The cells morphology was observed by inverted microscope, and the growing state of the third passage was detected by the cell counting meter of Vi-CELL. The cell surface markers CD105, CD14, CD73, CD34, CD44, CD45 and CDg0 were identified by flow cytometry. The expression of CK19, Oct-4, Nanog and vimentin was detected by immunocytochemistry, and the expression of c^-smooth muscle actin（ct-SMA） was tested by immunofluorescence. The differentiated potential of fibroblasts of the third passage into adipogenic, osteogenic and chondrogenic lineages was assayed. Results The primary passage fibroblasts showed the shape of spindle shaped or irregular polygon with a radiated or circinate of growing arrangement. The growth curve showed the ceils growth was slow on the first and second day, and quick during the third to fifth day, which reached platform stage on the sixth or seventh day. The fibroblasts highly expressed mesenehymal stem cell surface markers-CD73, CD105, CD44, CD90, but not expressed hematopoietic stem cell surface markers-CD14, CD34, CD45 by flow cytometry. And positive expression of vimentin, Oct4 and negative expression of CK19 were detected by Immunoeytochemistry. Positive expression of a- SMA was also detected by immunofluorescence. Muhidirectional differentiation induction indicated that the third passage cells could differentiate into adipogenic, osteogenic and chondrogenie lineages. Conclusions Human hypertrophic scar-derived fibroblasts show the biologic characteristics of mesenchymal stem cells, which may play an important role in wound healing and hypertrophic scar formation.
出处
《中华整形外科杂志》
CAS
CSCD
北大核心
2013年第4期273-279,共7页
Chinese Journal of Plastic Surgery
基金
国家自然科学基金（81060157）
贵州省优秀科技教育人才省长资金（黔省专合字2010-70）
