摘要
目的建立稳定的人肉芽组织成纤维细胞体外分离、培养体系,并探讨其生物学特性,为进一步研究成纤维细胞在人创面愈合中的作用提供参考。方法采用机械法和酶消化法分离培养人创面肉芽组织成纤维细胞,倒置相差显微镜下观察细胞形态及增值情况;分别绘制第3、5、10代细胞生长曲线。应用流式细胞仪(FCM)检测第3代细胞表面标志CD105、CD73、CD90、CD44、CD34、CD45、CD19、CD11b及HLA-DR的表达。取第3代细胞行免疫细胞化学检测vimentin和CK19的表达情况。结果原代培养出的成纤维细胞呈短梭形、多角形或不规则形;体外培养连续多次传代后获得形态较均一的成纤维细胞,以梭形为主,细胞呈放射状排列;细胞增殖良好,第3、5、0代细胞增殖能力无明显差异,有明显的指数生长期,体外能长期培养存活,并保持不分化状态。流式细胞仪检测细胞高表达CD105、CD73、CD90、CD44等间充质干细胞表面标志,不表达CD34、CD45、CD19、CD11b、HLA-DR等造血干细胞表面标志。免疫细胞化学检测vimentin阳性表达,CK19阴性表达。结论建立了稳定的人创面肉芽组织成纤维细胞体外分离、培养体系,获得的成纤维细胞表现出间充质干细胞的CD分子表型,有可能在皮肤创面修复中发挥重要作用。
Objective To establish a stable isolation and cultivation system of the human granulation tissue fibroblasts in vitro, and to investigate their biological properties, in order to further research its reference role in wound healing. Methods Fibroblasts were isolated and cultured from human granulation tissue by mechanical and enzymatic digestion method. The cell morphology and proliferation were observed under inverted phase contrast microscope. The passage 3rd,Sth,10th cells growth curve were drawn respectively. The third passage fibroblasts surface marker （CD105,CD73, CDgO,CD44,CD34,CD45,CD19,CD11 b, HLA DR） were identified by Flow Cytometry. And the expression of vimentin and CK19 were detected by immunocytochemistry. Results Primary cultured fibroblasts were short spindle, polygonal and irregular in shape. The morphology of fibroblasts were uniform by repeatedly passage cultured in vitro and showed spindle shaped. The proliferative capacity of the passage 3rd, 5th, 10th cells were no significant difference, and there is the exponential growth phase in long term survived. All fibroblasts highly expressed mesenchymal stem cell surface markers CD105, CD73, CD90, CD44, and no expressed hematopoietic stem cell surface markers CD34, CD45, CD19, CD11b, HLA-DR. The vimentin positive expression and CK19 negative expression were detected by immunocytochemis- try. Conclusion We establish a stable isolation and cultivation system of human granulation tissue fibroblasts in vitro. fibroblasts exhibit a mesenchymal stem cell phenotype may play an important role in skin wound repair.
出处
《西部医学》
2012年第12期2253-2257,共5页
Medical Journal of West China
基金
国家自然科学基金(编号:81060157)
贵州省优秀科技教育人才省长资金(黔省专合字2010-70)
