成年小鼠原代皮肤成纤维细胞的分离培养及其生物学特性

摘要

Objective:To establish time and cost saved method for culturing adult mice primary skin fibroblasts,and to explore its biological characteristics by immunofluorescence assay.Methods:The back skin,tail tip skin and ear skin were striping from adult mice(8～12 weeks),we chose two different DMEM high glucose extracts,three methods including tissue adherence method,enzyme digestion method and enzyme digestion tissue adherence method to culture adult mice primary skin fibroblasts.Evaluate those methods by microscopic observation through the cell numbers,the cell status,the length of culture cycle and cells′ purity.The biological characteristics of the cells were identified by immunofluorescence,CCK-8 and flow cytometry.Results:When using tissue adherent method to culture primary fibroblasts of adult mice's back skin,the Gibco fetal bovine serum(FBS) has little cell migrate out,the CLARK FBS has much more cell migration.The back skin was chosen to compare the different culture methods.When using enzyme digestion method,a very small amount cells were received and it has a terrible status,the tissue adherence method can provide more cells,but it took long time for the primary cell to migrate out.The method combining the enzyme digestion and tissue adherence can provide more cells and shorten the culture cycle.About the purity of cells,back skin was higher than that of the tail tip skin,the tail tip skin was higher than the ear skin.The proliferation curve of primary skin fibroblasts was presented a typical "S" type.Compared with the control group,UVB irradiation increased the apoptosis rate by 17％.Conclusion:The optimal method for primary skin fibroblasts culture is the Clark grade fetal bovine serum total extracts,adult mice back skin extraction,and enzyme digestion tissue adherence method.This combination can provide a large number of cells,shorten the culture cycle,reduce the cost.%目的:建立一种周期短、成本低的成年小鼠原代皮肤成纤维细胞分离培养方法,并探索其生物学特性.方法:取8～12周龄BALB/c小鼠背部、尾尖、耳部皮肤,配制2种血清的细胞培养液,采用组织块贴壁法、酶消化法、酶消化组织块贴壁法进行原代皮肤成纤维细胞的培养,通过显微镜观察比较原代细胞的数量、形态、培养周期及纯度;通过免疫荧光、CCK-8、UVB辐照、流式细胞术进行生物学特性鉴别.结果:背部皮肤组织块贴壁使用Gibco胎牛血清培养7d无细胞游出,CLARK特级胎牛血清细胞游出较多.背部皮肤经酶消化法得到细胞贴壁少;经组织块贴壁法细胞生长慢,培养周期长;酶消化组织块贴壁法细胞游出速度快、数量多、呈长梭形.尾尖取材量少,得到细胞少;耳部皮肤取材方便,但细胞纯度低.CCK8增殖曲线呈S型;相较于对照组,UVB辐照后细胞凋亡率增高17％.结论:CLARK特级胎牛血清、背部皮肤取材、酶消化组织块贴壁法是培养成年小鼠原代皮肤成纤维细胞最优的方案,可增加细胞得量、缩短培养周期,降低成本.