Immortalization And Characterization Of A Bovine Mammary Epithelial Cell Line

The bovine mammary epithelial cell is a secretory cell of mammary gland, and its cell number and secretory activity determine milk production. The Bovine mammary epithelial cell cultured in vitro is an effective reagent for studying the mechanism of mammary development and lactation. There is not existed a recognized bovine mammary epithelial cell line in the research area. Therefore, the study to establish an immortalized bovine mammay epithelial cell line is continuous. In this study, the research wok to immortalize and character a bovine mammy epithelial cell line was separated into4parts:1. The Immortalization of bovine mammary epithelial cell line. In our previous work, the establishment of a primary bovine mammary epithelial cell line, which has normal morphology, growth characteristics, and secretory characteristics, lays a foundation for cell immortalization. In this work, the immortalized bovine mammary epithelial cell (CMEC-H) was produced from obtained primary cells by the stable introduction of SV40large T antigen using a retrovirus. Four immortalized bovine mammary epithelial cell colonies were obtained by retroviral mediation, but only a single colony surpassed the50-passage mark and was designated the CMEC-H;2. Biological character of immortalized bovine mammary epithelial cell line. The telomerase expression of CMEC-H has consistently demonstrated the presence of telomerase activity as an immortalized cell line. The immortalized mammary epithelial cells grew in close contact with each other and exhibited the typical cobblestone morphology characteristic with obvious boundaries. The population growth rate of CMEC-H at passage5or50doubled in number within36h. The typical and representative karyotype of CMEC-H was counted at100chromosomes and more than the primary mammary epithelial cells. The immortalized cell line never induced tumor formation in nude mice, which suggested CMEC-H is not a cancaer line.3. Cell marker expression of immortalized bovine mammary epithelial cell line. For characteristic protein expression, the human breast cancer cell line MCF-7was used as a control for comparison and analysis. Cytokeratin7(CK7), CK8, CK18, and vimentin were localized primarily in a perinuclear and cytoplasmic distribution in nearly all MCF-7, which were identified by previous studies. CMEC-H expressed epithelial (cytokeratins CK7, CK8, CK18, and CK19), mesenchymal (vimentin), and stem/progenitor (CD44and p63) cell markers. In normal growth state, CMEC-H displayed epithelial stem/progenitor features.4. Cell differentiation of immortalized bovine mammary epithelial cell line. The hallmark of differentiated mammary epithelial cells is a secretion of milk-specific components regulated by lactogenic hormones. The relative expressions of milk proteins and synthesis-regulated genes:αs1-casein (CSN1S1), as2-casein (CSN1S2), β-casein (CSN2), κ-casein (CSN3), butyrophilin (BTN1A1), acetyl-CoA carboxylase-a (ACACA), fatty acid synthase (FASN), and peroxisome proliferator-activated receptor-y (PPARG) in immortalized epithelial cells compared to primary cells were detected to identifying the secretion ability of mammary cells by qRT-PCR. The transcription levels of CSN1S1, CSN1S2, and CSN2in CMEC-H were not significantly lower than the primary mammary epithelial cells. And the expression of CSN3significantly increased in immortalized cells compared with primary cells. The expression of PPARG was remarkably higher in immortalized cells than in primary cells. However, the expressions of BTN1A1, ACACA, and FASN in immortalized cells were lower than in primary cells. But the expression difference in all these genes between immortalized and primary cells could not be clearly detected by agarose gel electrophoresis, which showed the sensitivity of qRT-PCR. The Western-Blotting assay exhibited the same results. The induced expression of milk proteins, αs1-casein, β-casein, κ-casein and butyrophilin, indicated that CMEC-H maintained the synthesis function of the mammary epithelial cells.In summary, the established immortalized bovine mammary epithelial cell line CMEC-H is capable of self-renewal and differentiation, and can serve as a valuable reagent for studying the physiological mechanism of the mammary gland.