人体皮肤成纤维细胞的快速分离及多向分化能力鉴定

Isolation and identification of multidirectional differentiation of human skin fibroblasts

目的 建立快速分离人皮肤成纤维细胞的方法,并探讨成纤维细胞在成脂、成骨、 成软骨和成神经的多向分化潜能。方法 利用组织块培养法分离人体皮肤成纤维细胞,通过形 态学观察、流式分析、Vimentin 蛋白染色鉴定成纤维细胞;再利用生长曲线、核型分析、线粒体 染色分析不同传代细胞的增殖速度,线粒体及染色体形态的改变;最后进行成纤维细胞成脂、成 骨、成软骨和成神经的诱导分化实验,鉴定其多向分化潜能。两代细胞增殖速度及线料体相对 量的比较采用 t 检验统计分析。结 果 分离的皮肤成纤维细胞呈典型梭状及多角形;高表达细 胞表面标记物 CD90(NCF1,NCF2占比分别为 99.9,98.7)和 CD73(NCF1,NCF2占比分 别为98.2,85.6),但极少表达造血干细胞标记物 CD34(NCF1,NCF2占比分别为 1.8,2.6);细胞 Vimentin 蛋白表达呈阳性,阳性率为100;对细胞生长曲线进行分析,表明分离 后不同代次细胞增殖差异无统计学意义(t=1.586,P =0.1567);线粒体相对含量统计分析,同一株细胞系第5代(相对荧光强度值: 6876±577.8)与第 10 代(相对荧光强度值:7371±471.9)之 间的差异无统计学意义(t = 0.664,P = 0.543);核型分析分别显示传代后保持染色体数目为正常 46 条且形态无明显异常;经诱导后成纤维细胞可向成脂、成骨、成软骨和类神经分化。结论 利 用组织块培养法分离出的人体皮肤成纤维细胞状态稳定,增殖能力强,具有成脂、成骨、成软骨 和成神经多向分化诱导潜能,为细胞移植修复骨损伤、软骨损伤和神经损伤性疾病的临床研究 提供细胞来源及实验依据。

Objective To efficiently isolate human skin fibroblast cells with rapid proliferation and to investigate the multi-lineage differentiation potential of fibroblasts into adipogenic,osteogenic,chondrogenic and neurogenic directions.Methods The human primary fibroblast cells were isolated and cultured from the skin tissues.Morphological structures,flow cytometry and vimentin staining were used to identify the fibroblasts.Growth curves analysis,mitochondrial staining and karyotype analysis were used to identify the cell state of different generations.And finally fibroblasts were induced to adipogenic,osteogenic,chondrogenic and neurogenic directions.The difference between cell proliferation rate and the relative amount of mitochondrial was compared by t-test.Results The isolated fibroblasts showed a typical spindle-like morphology.Flow cytometry analysis showed that the isolated cells were highly express fibroblast surface markers CD90(NCF1,NCF2 accounted for 99.9﹪,98.7﹪,respectively)and CD73(NCF1,NCF2 accounted for 98.2﹪,85.6﹪,respectively),rarely express hematopoietic stem cell marker CD34(NCF1,NCF2 accounted for 1.8﹪,2.6﹪,respectively).All the fibroblasts positively express vimentin,the positive rate was 100﹪.The results of growth curves analyse showed that there was no significant difference in cell proliferation rate among multiple passages(t=1.586,P=0.1567).The relative content of mitochondria showed no significant difference(t=0.664,P=0.543)between the fifth generation(relative fluorescence intensity:6876±577.8)and the 10th generation(relative fluorescence intensity:7371±471.9)of the same cell line.Karyotype analysis showed that the number of chromosomes was 46 and the morphology of chromosomes were similar during passage.The multi-lineage differentiation assay showed that the fibroblasts could express adipogenic,osteogenic,cartilaginous and neurogenic differentiation markers.Conclusion Isolated fibroblasts have adipogenic,osteogenic,chondrogenic,neurogenic multi-directional differentiation potential,which can meet the needs of preclinical studies and provide cell sources for bone trauma,cartilage injury and neurodegenerative diseases.