Influence Of Digestive Enzymes、FBS And Prolactin On Cuituring The Bovine Mammary Epithelial Cell

This study explored the critical factors that influence the cultivation of bovine mammary epithelial cells. The results laid foundation for our further experiment about influence of nutrition factors on mammary gland cell milk protein synthesis. Breast tissue from Holstein dairy cattle in lactation period was used.1. Influence of time that tissue soaking in75%alcohol and different combination and concentration of digestive enzymes when isolating epithelial cells from bovine mammary tissue were studied, different concentration of fetal serum (FBS) which culture the cells was also studied. Tissues of similar size were soaking in75%alcohol for Os,30s,60s,90s,120s, after digestion and separation, the cell were cultured and observed. The results showed that cells harvested from tissues soaking in75%alcohol for60s had the best growth conditions. In our study, the effect of0.5%collagene Ⅰ,0.5%Ⅱ collagene,0.2%collagene Ⅰ.0.2%collagene Ⅱ,0.25%trypsin+0.5%collagene Ⅰ,0.25%trypsin+0.5%collagene,0.25%trypsin+0.2%collagene Ⅰ, and0.25%trypsin+0.2%collagene Ⅱ in the digestion of mammary gland tissue were compared. After2h of digestion, cells harvested from0.25%trypsin+0.2%collagene Ⅱ digestion show the best growth state. The concentrations of FBS in the cell medium were as follow:0%,5%,10%,15%, and20%. Results showed that the cell number have no significant difference (p>0.05) when cultured in5%and10%FBS medium,which were significantly higher than in0%,15%, and20%FBS medium(p<0.05).2. Different time to add prolactin (PRL) on the morphology, activity and gene expression of bovine mammary epithelial cell were studied. Prolactin was added in2nd generation or3rd generation cells either from the beginning or24h before harvest. The results showed that the morphology and vitality between the2nd generation and3rd generation cell had no significant differences (p>0.05). mRNA level of asl-casein, K-casein, and β-casein gene were significantly higher in the2nd generation cells which added PRL24h before harvest when compared with groups with no prolactin addition or added prolactin from the begining (p<0.05). The3rd generation cell cultured with different prolactin addition ways had no significant (p>0.05) different in expression level of asl-casein, K-casein, and β-casein gene.