原代成人皮肤真皮成纤维细胞系的构建

Isolation,Culture and Identification of Primary Human Skin Dermal Fibroblasts Cell Lines

目的:对人皮肤成纤维细胞进行分离、纯化、培养及细胞鉴定,探讨优化的分离及纯化方法,为构建表皮-真皮皮肤模型提供种子细胞。方法:使用两步消化法提取人真皮层,酶消化法提取成纤维细胞后培养数代,对细胞进行形态学观察,HE染色,免疫细胞化学鉴定细胞标志物Vimentin,MTT法测定细胞增殖,流式细胞仪检测细胞凋亡情况。结果:利用倒置显微镜及HE染色,可见细胞为散在分布的壁细胞,细胞汇流时呈鱼群样或漩涡状排列,细胞在30代内形态保持不变;细胞增殖曲线大体呈S型,流式细胞仪测得细胞凋亡率分别为(8.15±0.618)%和(7.83±0.415)%。结论:优化提取真皮成纤维细胞的方法,构建2株不同年龄的真皮成纤维细胞系,为药妆研究以及重组皮肤模型的研究奠定了基础。

Objective：In order to discuss the isolation methods of optimization, this experiment isolate, culture and cell identify the adult human dermal fibroblasts successful and provide the seed cells for epidermal -dermal skin model. Method：The dermal was harvestd by two- step enzymatic digestion,and the fibroblasts were isolated by enzymatic digestion with 0. 5% lV collagenase and cultured for passa- ges,then begin to implement cell morphological observation, HE staining, cell immunohistocbemistry to identify Vimentin as the cell mark- er, and MIT assay for proliferation. Result：The fibroblasts was spindle shaped adherent cells dispersed which observed with inverted mi- croscope and HE stain. When the cells tended to confluence, they unfolded fish - school configuration or eddy - like configuration and the flbroblasts stay the morphology unchanged under 30 passages,the growth curve demonstrates S, shaped mainly, the cell apoptosis rates are （ 8. 15 ± 0. 618 ） % and （7. 83± 0. 415 ） %. Conclusion： Optimizing the isolation methods of yielding dermal fibroblasts successfully, and established three dermal fibroblasts cell lines with different ages, finally laying foundation for the research of cosmeceutical and reconstruc- ted skin model.