| In vitro cell culture was a fast and easier method to regulate cell growing by simutating in vivohormone environment and structure, and could facilitate the research on cell growing capability indifferent culture conditions. Mouse was a great donor for mammary gland research on cell levelbecause it was easily to be gotten and maneuverability. Since nowaday, there was not an unitedoperational standard to regulate the isolation and culture of mammary epithelial cell, which leadedto unrepeatable results, to improve isolation, culture technology and to set a new and perfect invitro culture system became two most important tasks in the fields of mammary development andfunction researching.Heregulin-α(HRGα) was a regulatory polypeptide having distinct biological effects, such asgrowth stimulation, differentiation, invasiveness and migration in mammary epithelial cells. Itplayed a role in the morphogenesis and ductal migration of mammary epithelial cells, promoted theresponsiveness of these cells to lactogenic hormones in vitro and induced the differentiation ofmammary epithelium into secretory lobuloalveoli.The aim of this research was to set up a complete in vitro culture system, in which to discussthe influence of HRGαon the proliferation and metabolism of mouse mammary epithelialcells (MMECs), to offer important academic evidences for the further research on morphology,structure and functions of mammary epithelial cells, and to reveal the role of HRGαin the course ofmammary gland development. And finally to provide feasible technique and belivable acadimicsupport for raising the quality and quantity of milk production by enhancing mammary glanddevelopment and its functions in the way of artifical regulation.Collagenase one step digestion method, trypsin and collagenase sequential digestion methodwere applied seperately to obtain MMECs, and the two methods were compared according to themorphology of cultured cells. Results showed that trypsin and collagenase sequential digestionmethod was helpful to produce abudance and high purity mammary gland epithelial cells withuniform morphology. Simple and easy to operation were characteristics of upper method.In this research, the effects of pH and concentrations of serum in medium on the growing ofMMECs were checked. Results showed that pH7.4 and 10% serum supplying was most suitablemedium for culture. There were significant difference of the number of cell in pH6.8, 7.6 andpH7.2, 7.4 groups(p＜0.01), In pH7.2 and pH7.4 groups, significant difference was notfound(p＞0.05). When 5% FBS were supplied, significant difference were found among allgroups (p＜0.01), 10% and 15% FBS groups no significant difference were found (p＞0.05).Phase contrast microscope and MTT assay were performed to distingush the biological characteristics of MMECs in different stage. The results demonstrated that mouse mammary glandin gestation was the best period for cell isolation because in this stage the capability of survive andproliferation of cell was better than that in the stage of lactation.Immunohistochemistry and RT-PCR were applied to identify keratin-18, an important symbolof cell andβ-casein, a specific seretion protein. The results demonstrated that obtained cell wasmammary epithelial cells with normal milk giving function which illustrated that MMECs in vitroculture system had been constructed successfully.On the basis of the new in vitro culture system of MMECs, the effect of HRGαon MMECswas preliminary studied. Results showed that suitable amount of HRGαcould promote proliferationand metabolism of MMECs significantly. 0.1～20ng/ml HRGαcould promote MMECs proliferationand increased the amount of total protein and lactose(p＜0.05), 50ng/ml and 100ng/ml HRGαinhibited MMECs proliferation, and lowed the contents of total protein and lactose (p＜0.05).
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