Establishment Of Dairy Cow Mammary Gland Epithelial Cell Line

Mammary gland epithelial cells are specialized secretory function cells, and are also the all foundation of lactation function materials for studying on lactation starting, functions maintenance, degradation etc, physiological process internal and external cytokine regulation, signal transduction, function genes expression and the formation of mammary gland tumors, canceration. According to nowaday studies, between ruminants and human, lactation control mechanism existing a great differences. Compared to other species of animals, mammary gland regulation mechanism of ruminants has not been fully proved, especially that the culture of lactation dairy cow mammary gland epithelial cells which have high level secretion of milk is difficult.Dairy cow mammary gland epithelial cells cultured in vitro can be used for research of mammary gland development, lactation mechanism, and biological reactor applications etc. Although there have been some reports on cultured dairy cow mammary gland epithelial cells, researchers always can not succeed in establishing dairy cow mammary gland epithelial cells line, cells gained are unsuited for the research on lactation mechanism and detection of mammary gland expression vector. Establishment of dairy cow mammary gland epithelial cell line is fairly good as an external model for the establishment of transgenic animals, study of the lactation quantity improvement, and also auxanology, mastopathy neo-Confucianism and bioengineering etc. Therefore, it is necessary to culture dairy cow mammary gland epithelial cell in vitro and study the role of hormones on mammary gland epithelial cell function.This study selected slaughtered health Holstein dairy cow mammary gland tissue of late stage lactation as experimental material, and used tissue block inoculation method to culture dairy cow mammary gland epithelial cells successfully, cytokeratin 18 was used for immunofluorescence staining of mammary gland epithelial cells. According to cells inoculation survival rate, population doubling time, growth curve morphology and other biological characters of detection, a dairy cow mammary gland epithelial cell line was established. In addition, lactose and casein protein contents of dairy cow mammary gland epithelial cells were measured by HPLC method, restively reflecting effects of the hormone on lactating dairy cow mammary gland epithelial cells function, thus this study gave important basis for dairy cow mammary gland epithelial cells bioreactor model and study of lactation mechanism.The experimental results showed that dairy cow mammary gland epithelial cells had been purified, and the establishment of dairy cow mammary gland epithelial cell line had been identified. Through passage of the mammary gland epithelial cells and repeatedly frozen and recovery, the mammary gland cell line was proved to have achieved long-term preservation, and a large number of mammary gland epithelial cells were gained. Dairy cow mammary gland epithelial cells grew well in the culture liquid medium after adding with epidermal growth factors, and had good characters of proliferation, and the cells over 20 passages of proliferation remained excellent activity contents of casein protein of mammary gland epithelial cells cultured with insulin remained little changed, prolactin and progesterone group significantly increased. For three hormones in the culture medium, casein protein contents at different time points, the results showed that the contents of casein increased significantly within 24 hours. The results of hormones on the cell lactose contents by HPLC showed that there was little impact of insulin on cells lactose contents, while prolactin and progesterone significantly increased. The role of three hormones in the culture medium at different time points on lactose contents showed that concentration of the lactose by insulin was rather high, and changed little, but for prolactin and progesterone groups in all of the time the contents were not obvious changed and remained lower. Studies also were done to find their effects on the cell viabilities by three hormones at different time points, prolactin and progesterone both increased, while insulin was not obvious. The results suggested that insulin could maintain lactose synthesis of dairy cow mammary gland epithelial cells, while prolactin and progesterone both helped casein protein synthesis. The results suggested that these three hormones were essential to maintain lactating function for cultured dairy cow mammary gland epithelial cells.