人皮肤成纤维细胞原代培养及生物学特性

Primary Culture and Biological Characteristics of Human Skin Fibroblasts

目的探索和建立人皮肤成纤维细胞原代培养的方法,为皮肤成纤维细胞在临床医学中的应用提供可靠的科学依据。方法胰蛋白酶消化法分离培养人皮肤成纤维细胞;细胞生长曲线及MTT法测定细胞增殖能力;流式细胞仪测定细胞生长周期及细胞增殖指数;倒置显微镜下观察成纤维细胞生长状态;HE染色观察细胞爬片下的形态及着色;免疫组织化学染色观察成纤维细胞中间丝波形蛋白;电子显微镜下观察成纤维细胞超微结构。结果体外用胰蛋白酶消化法建立了人皮肤成纤维细胞;传5代内细胞及传5代内冻存复苏后人皮肤成纤维细胞增殖能力均很强;倒置镜下观察、HE染色、波形蛋白免疫组化染色及电镜下观察鉴定培养细胞的形态及生物学特性符合成纤维细胞。结论本研究确立了体外人皮肤成纤维细胞原代培养。

Objective To establish a method for primary culture of human skin fibroblasts and to provide a reliable basis for clinical application of skin fibroblasts.Methods Human skin fibroblasts were cultured with trypsin digestion.The proliferation of fibroblasts was detected by growth curve and MTT assay.The cell cycle and proliferation index were determined by flow cytometry.The morphology and ultratructure of the fibroblasts was observed under inverted and electron microscope,respectively.Immunohistochemical staining was performed to observe the vimentin in fibroblasts.Results Human skin fibroblasts were obtained.The proliferative ability of the fibroblasts was high within 5 passages or after cryopreservation.The morphology and biological characteristics of fibroblasts were confirmed under inverted and electron microscope and by HE and immunohistochemical staining.Conclusion The method for primary culture of human skin fibroblasts in vitro is successfully established.