To establish the isolation and culture system of Qinchuan cattle somatic cells,this study used three different methods to ex-tract 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 diges-tion 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.
维普
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