| Mammary tissue of dairy cattle has the special function of synthesis and secretion,and this function is accomplished by mammary epithelial cells. The number andsecretion activity of mammary epithelial cells affect directly the milk yield of dairy cows.It was known that the cow breast tissue has remodeling function. Breast tissuedegradation and remodeling were accompanied with lactation period in cycles. Duringthe process of remodeling of breast tissue, if the number of epithelial cells with thefunction of lactation increased more and the milking activity is stronger in the limitedspace, which has the vital significance to the milk yield. It was demonstrated that therewas sensitive cell populations that contains abundant breast stem cells for Wnt protein inadult mouse mammary glands, and confirmed that Wnt3a protein had obviously role forpromoting colony formation on breast stem cell and maintaining the function forfunctional glands formation. It was inferred that the activation of Wnt pathway canpromote proliferation of cow mammary epithelial cells.In the present study, healthy cow breast tissue was selected as raw materials.Mammary epithelial cells were cultured by tissue culture method and observed byinverted microscope. The results showed that: on4d, fibroblasts with loose space firstlygrew out from mammary tissue. Eepithelial cells, paving stone-like and cobblestone-like,were present about6d. The nucleus with2～4nucleolus was large and obvious.Improvement of β-casein extraction. Because of mammary epithelial cells wereprimary single-layer adherent cells, cell numbers were limited. Therefore, it was difficultthat the secretion of MEC in the culture medium was detected. Four different methodswere used to extract beta casein from cell culture medium, and the extract was identifiedwith western blot. A new extraction method forβ-casein was established firstly. Thismethod, that is physical precipitation, was simple, economical and practical. And themammary epithelial cells with normal secret capacity were proved. Thereby, a bovinemammary epithelial cell culture system was established initially.Different content exogenous recombinant Wnt3a protein (10ng/mL,25ng/mL and50ng/mL) was added into culture medium when the medium was replaced for the firsttime at48h. Cultured cells on7-11d of the four groups were selected (two repeat) andpurified by enzyme digestion method. Then, the epithelial cells were counted and growth curve was made. Culture solution on10,11and12day was collected to detecting betacasein by Western blot. At the same time, fibroblasts were removed by enzyme digestionfor collecting the purified epithelial cells. Then total RNA of epithelial cells wereextracted. The expression of Frizzled1receptor and beta-catenin mRNA in Wnt/beta-catenin signaling pathway were detected by fluorescence quantitative PCR. The resultshowed that: Addition of10ng/mL,25ng/mL and50ng/mL Wnt3a protein into culturemedium, the relative expression of Frizzled1receptor is0.79、1.13and1.89separately,and the relative expression of beta-catenin is0.44、0.91and1.19separately.Conclusion: The culture system of cow mammary epithelial cells was establishedpreliminary, and a new extraction method of beta casein was explored successfully;50ng/mL Wnt3a was the optimum concentration to stimulate mammary gland epithelialcells. The epithelial cells number and secretion of beta casein were significantlyincreased than that of other groups. The expression of Frizzled1receptor mRNA, whichis the key receptor in classic Wnt signaling pathway, increased obviously. Significantvariation of the expression of beta–catenin was not found. |
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