Establishment And Application Of The In Vitro Culture System Of Bovine Mammary Epithelial Cells(BMEC)

Culture system was established to study the effect of linoleic acid (LA) and vitamin A (retinoic acid,RA) on the proliferation and differentiation of Bovine mammary epithelial cells (BMEC).The first part examined a comparative study that was conducted on BMEC isolated from mammary gland explant, mechanical fragmentation, assimilation of collagenase and centrifugal method from milk based on their morphology.The BMEC were incubated with the culture media containing DMEM/F12(V/V=1:1) and 20% fetal calf serum (FCS), 5μg/ml insulin(I),1μg/ml hydrocortisone(HC), 1μg/ml progesterone,100IU/ml penicillin,100IU/ml streptomycin,5μg/ml transferrin and so on. Cells were routinely cultivated in Petri-dishes attached with collagen gels. Cells were maintained at 37℃in an incubator with , in a humidified atmosphere containing 5% CO2. The major morphology of epithelial cells are characteristic guboidal and cobblestone. The phase contrast photomicrographs indicated the presence of domelike and nipple structures in the proliferated cells with the phenomenon of lactescence. The BMEC from milk proliferated faster than those isolated from mammary gland explants; and had no potential problems of fibroblast outgrowth. The BMEC from milk began to proliferate within the 4th-6th days of the culture, and on the 15th day the cells covered the basement membrane of the plastic dishes completely. Constantly the BMEC proliferated from mammary gland tissue began to grow after two weeks of the culture and covered the basement membrane until 30th days. The results showed that milk can alternatively be an effective source for isolating and culturing of BMEC.The second part established the culture system of BMEC .Firstly, the cell biological properties were observed and investigated from the cell planting survival efficiency, cell population doubling time and cell growth curve. The results showed that the BMEC grows well and proliferates vigorously in this culture system.There was a significant difference in the diameter of cells isolated from mammary gland explant and bovine milk(P<0. 001). The results for immunochemical identification of cytokeratin-18, the SDS-PAGE and amplification ofαs1-casein gene in BMEC indicated that accomplished cells are BMEC.The third part examined the effect of LA or RA on BMEC.MTT assay was used to examine the activity of BMEC. In in vitro experiment,the BMEC were cultured in DMEM/F12 containing 0(control),10,50,100,200,400uM LA, and 0(control),0.15,1.5,15,150,1500,15000ng/ml RA. The activity of BMEC with 400uM LA or 1.5 ng/ml RA were significantly lower than that the control, and caused a greater inhibition effect.