Culture Of Mammary Epithelial Cells And Identification Of Mammary Stem Cells

Dair y Cow mammar y epithelial c ells(DCMECs) cultur e system as a commonly tool used in exper iments can provide model for the res earch of development of cow mammary and f or the regulation of lactation and mammary gland bior eactor. The establis hment of immort alized DCMECs line has not yet mature, in order to gain cells which has the lactation function or to build three-dimens ional model of the ac ini we need to induce and s imulate phys iologic al conditions and the pr imary culture. Mammar y gland is an organ that develops af ter born, mammar y gland develops rapidly in the adolescence and pregnancy which related w ith the self-renewal and differentiation of mammary gland stem cells(Ma S Cs). Research shows that there is a certain proportion of stem cells in mammary gla nd, but the specif ic molecular mar kers us ed to identify the stem cells is still not ver y clear. Some substances such as hor mones and growth f actors can promote the development of breast and regulate the lactation in vivo, promote the prolif eration of mammary epithelial cells, inf luent the adhes ion, migr ation, extend and differ entiation of cells and regulate the Ma SCs in vitro. The inf luence of hor mones and gr owth factors on the expans ion, differentiation and plur ipotency of Ma S Cs and the r egulation of mamm ary epithelial cells’ growth, differentiation or the built of thr ee-dimens ional model of the acini is also worth in- depth research.This study uses pr imary cultured DCMECs as the exper imental model, to explore the method of gaining a lot of mammar y epithelial cells in a short time, s elect new spec if ic molecular mar kers used to identif y the stem cells, observe the effects of xanthos ine and IGF- Ⅰ on epithelial cells and stem cells. Us ing collagenase digestion in vitro to cultur e cow mammar y gland epithelial cells, by draw ing cell curve, detecting kar yotype and casein, use q RT-P CR to detect the potential lactation of pr imar y and passage cells; by laser Confocal, Br du label-r etaining test and the way of cell division to identify stem cells in mammary epithelial cells cultured in vitro.Results showed that:(1) Us ing digestion mixture cons ists with collagenase Ⅳ, hyalur onidase and DNas eⅠcan receive lots of dairy cow mammary gland epithelial cells in 3h. The results show that the pr imary and pass age generation cells have 60 chromosomes and have typical epithelial cell morphology. They ar e CK18 pos itive cells which can synthes is and secrete β-casein, the cells can be used as experimental material in the further experiments.(2) In vitro cultur e, q RT- P CR detect the m RNA levels of pr olactin, growth hormone, insulin- like growth factor- Ⅰ and their receptors cell in P0, P3, P6, P9. Results dis played that hormone, growth factor and their receptors m RNA levels in pr imary and passage cells showed very signif icant differ ence(P<0. 01). DCMECs will r apidly los e some phys iological character istics and the ability of milk synthesis under the condition w ithout inducing substances.(3) We aimed to identify putative bovine mammary epithelial stem cells which were Brd U label retaining epithelial cells, to analys is the express ion and location of two new molecular markers(FNDC3B and PROCR) of Ma S Cs and test the feas ibility of using them to identif y the mammary epithelial stem cells. The r esults showed that the proportion of Brd U label-r etaining epithelial cells(LRECs) was near ly 0.4% af ter a 25 d continuous culture(passaged 4 times), a few of cells were pos it ive for F NDC3 B or P ROCR. Moreover, observing the Brd U labeled epithelial cells wer e asymmetr ic divis ion. But a certain percentage of mammary stem/pr ogenitor cells will be retained, whose potential effects on the r egulation of lactation and mammar y ac inar r emodeling are worthy of attention.(4) Make growth curve, add xanthos ine and IGF-I in logar ithmic phas e, detect m RNA levels of prolactin, growth hor mone, insulin- like growth factor- Ⅰ and their receptors and th-e levels of FNDC3 B and PROCR m RN A in primar y and passage cells. In P0-P9 cells, af ter adding xanthos ine and I GF-I the expr ess ion of P ROCR m RNA incr eas ed s ignif ic antly(P<0.05), the change of FNDC3 B m RNA express ion is not signif icant(P>0.05). The relativ e express ion of PRL/P RLR, GH/GHR, IGF-I/I GF-I R m RNA in P0-P3 cells showed a trend of rise, but in P6-P9 cells showed a downward tr end. It is suggest that xanthos ine and IGF-Ⅰ can promote the prolif eration of epithelial c ells and stem cells, and was positively reg ulate gene expression in early lactation of mammary epithelial cells.