双酶联合消化法培养人皮肤成纤维细胞

The Study on Culture of Human Skin Fibroblasts by Double Enzyme Combined Digestion

目的建立一种便捷、高效的人皮肤成纤维细胞体外培养技术,获得大量高纯度原代细胞。方法取健康成年男性包皮标本,去除皮下组织,将皮肤剪成小皮片,以0.25%的胰蛋白酶溶液4 ℃消化过夜后分离去除表皮;将真皮组织剪碎,加入0.1%胶原酶Ⅱ溶液后于37 ℃ CO2孵育箱中消化3~4 h,筛网过滤,收集滤液后离心、培养。绘制细胞生长曲线,HE染色观察细胞形态,细胞免疫荧光、Western blot检测Vimentin鉴定成纤维细胞,取第3代、第6代细胞以流式细胞术检测细胞纯度。结果获得原代人皮肤成纤维细胞;细胞生长周期:0~2 d为潜伏期,2~5 d为指数生长期,之后进入平台期;细胞为梭形或星形;Vimentin表达呈阳性,鉴定为成纤维细胞;第3代、第6代成纤维细胞纯度分别为95.82%、99.51%。结论利用双酶联合消化法成功培养高纯度原代人皮肤成纤维细胞,为进一步研究奠定基础。

Objective To establish a convenient and efficient in vitro culture technique for human skin fibroblasts,and to obtain a large number of high purity primary cells.Methods Foreskin specimens of healthy adult males were taken to remove the subcutaneous tissue.The skin was cut into small pieces,and the epidermis was separated and removed after digestion with 0.25% trypsin solution at 4 ℃ overnight.The dermal tissue was cut,added with 0.1% collagenase Ⅱ solution,and digested in a 37 ℃ CO2 incubator for 3 to 4 hours.After it was sieved,filtrate was collected for centrifugation and culture.Cell growth curve was drawn,and cell morphology was observed by HE staining.Vimentin was detected by cell immunofluorescence and Western blot to identify fibroblasts,and cell purity was detected by flow cytometry in the 3rd and 6th generation cells.Results The primary human skin fibroblasts were obtained;the cell growth cycle included the lag phase(0 to 2 days),the exponential growth phase(2 to 5 days),and the plateau phase;the cells were fusiform or star-shaped.Vimentin expression was positive,so the cells were identified as fibroblasts.The purity of the 3rd and 6th generation fibroblasts were 95.82% and 99.51%,respectively.Conclusion High-purity primary human skin fibroblasts were successfully cultured by double enzyme digestion method,which laid a foundation for further research.