Establishment And Characterization Of Yak Mammary Epithelial Cell Lines

The objective of this study was to establish a culture system of yak mammaryepithelial cell in vitro, in order to provide evidences for further studies on thedevelopment and its milking mechanism of yak mammary gland at the cellular level.Meanwhile, the cells can be used as nuclear donor cells for cloning yak. Themammary gland tissue of a lactational yak was used as the research material in thestudy as follows:(1) The primary cells of yak mammary gland were obtained by methods of tissueculture and collagenase dissociation, then enriched the epithelial and myoepithelialpopulation by selective detachment and attachment methods, at last the yak mammaryepithelial cell line (YMEC) and myoepithelial cell line (YMM) were established.YMEC cells could long-term maintain (35passages, about6months) in DMEM/F12medium supplemented with10%fetal bovine serum. YMEC cells with thecharacteristic cobblestone morphology of epithelial cells, twenty four hours afterplating, cells formed monolayer island, then formed lumen-like structures。Aftersubculturing25times, the cells differentiated into two shapes. YMM cells weremaintained for more than6months (40passages) in DMEM/F12mediumsupplemented with10%fetal bovine serum without obvious signs of senescence.YMM cells showed a distinctive stellate morphology and extended out filopodia fromthe cell body at ends. Moreover, YMM cells formed a monolayer but did not formcontiguous membrane-to-membrane contacts when grown to confluency. YMM cellschanged to polygonal-like in morphology after15passage.(2) In this research we studied some characteristics of YMEC cells whichincluded cell viability, growth curve, doubling time of cell population, chromosomalanalysis and ultrastructural characteristics. Meanwhile, the influence of hormone onmRNA expression of β-casein was studied. We also cultured YMM cells on collagenmembranes. At last YMEC cells were identified by immunocytochemistry andRT-PCR. The results showed that the growth curve of YMM cells conformed to therule of “S” sigmoid curve, with an estimated population doubling time of45-48h, maintained a normal diploid chromosome modal number of2n=60which contained29pairs of telocentric chromosomes and a pair of X. The result of sterility wasnegative. YMEC cells remained immunopositive to antibodies against cytokeratin,vimentin and immunonegative to antibody against α-smooth muscle actin. The surfaceof cells had numerous microvilli and the cytoplasm contained an extensive network ofrough endoplasmic reticulum and mitochondria. Furthermore, the mRNA expressionof beta-casein was detected by RT-PCR after cells stimulated by some hormones.When YMM cells cultured on collagen membranes could form structures of duct-likeoutgrowth and spherical mass.(3) In this research we studied some characteristics of YMM cells which includedcell viability, growth curve, doubling time of cell population and chromosomalanalysis. YMM cells were identified by Immunocytochemistry and oxytocinresponsiveness. The results showed that the growth curve of YMM cells conformed tothe rule of “S” sigmoid curve, with an estimated population doubling time of66-68hand reduced to approximately59-61h after that changed in morphology, maintained anormal diploid chromosome modal number of2n=60. The result of sterility wasnegative. Noticeably, YMM cells appeared oxytocin responsiveness whensupplemented oxytocin in medium. Furthermore, YMM cells remainedimmunopositive to antibodies against α-smooth muscle actin, cytokeratin7andvimentin.