Abstract: To establish the isolation and culture system of Qinchuan cattle somatic cells, this study used three different methods to extract fibroblasts from the ear margins of Qinchuan cattle. Results showed that fibroblasts could be obtained after 17 d of tissue block culture, while the double enzyme digestion method could be cultured for 9 d for passaging. Compared with that of the enzymatic digestion method Ⅱ（0.9×106/mL）, the concentration of fibroblasts obtained by the enzymatic digestion method I（1.24×106/mL）was high and the difference was significant. Third-generation fibroblast immunofluorescenceand flow cytometry identification results showed cell purity of over 95%. The purified fibroblasts were cultured until the logarithmic growth phase at 3 d, and the proliferation rate decreased at 8 d and entered the plateau phase. In conclusion, the dual enzyme digestion method I (0.25% trypsin digestion for 30 min and 1% collagenase for 60 min) was the best method for isolation and culture of Qinchuan cattle ear margin fibroblasts.

Key words: Qinchuan cattle, skin tissue, fibroblast, tissue culture method, enzyme digestion method