The Effect Of6-bromoindirubin-3-oxime On Biological Characteristics Of The Mammary Epithelial Cells In Goat

Mammary epithelial cells of the mammary gland need to go through a series of changesof biological characteristics in mammogenesis and lactation processes, including proliferation,differentiation and apoptosis. These changes are regulated by hormones, cytokines and so on.Related studies showed that Wnt signaling pathway may play an important role in theregulation of mammary gland development and lactation. However, after Wnt signalingpathway activation, if the biological characteristics of mammary epithelial cells was affected,remains to be further studied. BIO(6-bromoindirubin-3-oxime) is a Wnt signaling pathwayactivator, BIO can specifically inhibit GSK-3(glycogen synthase kinase-3), thereby activatingthe Wnt signaling pathway. In order to study the effect after activated Wnt signaling pathwayon biological characteristics of goat mammary epithelial cells, the present study isolated andin vitro cultured goat mammary epithelial cells，then add BIO into the medium, detected goatmammary epithelial cell morphology, the expression of marker protein cytokeratin18and cellbiological characteristics such as growth curves, and compared the difference. The resultswere as follows:1. The isolation, purification and identification of goat mammary epithelial cellThe mammary tissue was collected from healthy dairy goat lactation by asepsis surgery,and the goat mammary epithelial cells were isolated and cultured by tissue adherent culturewith tissue re-shift methods. Because of the primary mammary epithelial cells usually mixedwith a large number of fibroblasts, and the fibroblasts and the epithelial cells could be purifiedaccording to their different sensibility to trypsin, the mammary epithelial cells were isolatedand purified by different trypsin digestion duration. Purified epithelial cells arecobblestone-like, internal cell nucleus obviously, with2~4clear nucleoli, in line with themorphological characteristics of mammary epithelial cells. The cytokeratin18and β-caseinare protein markers of the mammary epithelial cells, which were detected in purified cellsindicating that cells were purified mammary epithelial cells. The results of the cell growthcurve detection in different cell inoculum density showed that the cell growth curves weretypical "S" shape, the growth incubation period of goat mammary epithelial cell is2to3days, afterwards the cells entered logarithmic growth phase, and at the6thday after inoculationentering into the plateau phase.2. The effect of6-bromoindirubin-3-oxime on biological characteristics of goatmammary epithelial cellsAdding2.5μmol/L BIO to goat mammary epithelial cell medium, some cells were nolonger adhered on bottom of the culture dish, the remaining adherent cells formed acinar-likestructure. The acinar-like cells had a high expression level of cytokeratin18. The cell growthcurve detecyion showed that the goat mammary epithelial cell proliferation were inhibitedafter adding BIO, indicating the formation of acinar-like structure is due to migration andassemble of cells, rather than cell proliferation.The results indicated that the combination method of adherent tissue and differentdigestion could get purified goat mammary epithelial cells. After the treatment of BIO, Wntsignaling pathway was activated, the goat mammary epithelial cells stopped proliferating, andformed a typical acinar-like structures.