成年小鼠背部皮肤角质形成细胞的原代培养及其与乳鼠角质形成细胞的对比性研究

Primary culture of keratinocytes from adult mouse back skin and comparison with neonatal mouse keratinocytes

目的:探索成年小鼠背部皮肤角质形成细胞有效且高生长率的原代培养方法;对比乳鼠和成年小鼠背部皮肤角质形成细胞的增殖、集落形成效率与凋亡能力。方法:取正常成年小鼠背部皮肤,分别利用4种方法分离其表皮与真皮,取表皮分离培养角质形成细胞;免疫荧光进行细胞鉴定;调整种板密度、种板方式观察细胞生长状况;利用CCK-8、EdU、结晶紫染色法和TUNEL染色检测乳鼠与成年小鼠背部皮肤角质形成细胞的增殖、集落形成效率与凋亡情况。结果:4种成年小鼠表皮-真皮分离方式中,二步消化法获得的细胞量最大、细胞增殖能力最强、集落形成效率高;水滴状种板法较普通平铺种板法集落形成率高;乳鼠角质形成细胞最适种板密度约为3.2×10^4/cm^2,成年小鼠背部皮肤角质形成细胞最适种板密度约为1.6×10^4/cm^2;与成年小鼠背部皮肤角质形成细胞相比,乳鼠角质形成细胞增殖能力较强、集落形成效率较高。结论:成功建立了较为完善的成年小鼠背部皮肤角质形成细胞原代培养体系。

AIM:To explore an effect and high growth rate of primary culture method for keratinocytes from the back skin of adult mice,and to compare the proliferation,colony formation and apoptosis of keratinocytes from the back skin of neonatal mice and adult mice.METHODS:The back skin of adult mice was digested by 4 different methods.The keratinocytes were isolated and cultured from the epidermis,and were identified by immunofluorescence staining.The density and the pattern of seed plates were adjusted.CCK-8 assay,EdU assay,crystal violet staining and TUNEL method were used to detect the proliferation,colony formation and apoptosis of the keratinocytes from the back skin of neonatal and adult mice.RESULTS:Among the 4 different methods of epidermal-dermal isolation in the adult mice,the two-step digestion method had the largest number,the strongest cell proliferation ability and the highest efficiency of colony formation of cells.The water-drop seed plate method had a higher rate of colony formation than the common seed plate method.The optimum density of keratinocytes in neonatal mice was about 3.2×10^4/cm^2,and the optimum density of keratinocytes in adult mice was about 1.6×10^4/cm^2.Compared with the adult mouse,the neonatal mouse back skin keratinocytes had stronger proliferation ability and higher colony formation efficiency.CONCLUSION:A primary culture system of keratinocytes from the back skin of adult mice has been successfully established.