Immortalization Of Bovine Mammary Epithelial Cell With Multipotency

Bovine mammary epithelial cells (BMECs) are perfect cells for bioreactor due to their secretion funtion. However it is difficult to culture BMECs for long time in vitro and the cell life span is limited. So it is very meaningful to get immortalized BMECs. There are many immortalization methods, such as transfection of SV40 large antigen, HPV E6 and E7, silence of anti-ongene, overexpression of ongene and so on. Recently, the hTERT got more and more attention because of its security, the cell transfected by hTERT could keep normality. The aim of the present study was to immortalize BMECs by hTERT. During immortalization, we found there were some bovine mammary stem cells (BMSCs) in bovine mammary epithelial cells. At the same time, we compared the BMECs with hTERT-BMECs for nuclear transfer embryos developmetal potential. We got the results as followed:(1) Bovine mammary epithelial cells had been successfully separated in the test, and the cells could be stained by Keratin. In vitro, the cells could secretβ-casein under induction condition.(2) The media added EGF or HGF can promote the bovine mammary epithelial cells proliferation,but when only 17β-E2 was added, there was no effect on the cells proliferation. When 17β-E2 and EGF were both added in the medium, 17β-E2 could enhance the effect of EGF on bovine MECs, but 17β-E2 has no synergistic effect with HGF.(3) After transfection with hTERT, the bovine mammary cell grew rapidly, but the cell still kept diploid karyotype, and no tumor formed by injection of the cells in nude mouse and the cells have telomerase activity. All of those domenstrated that the cells is immortalized cells.The cells could expressβ-casein under hormones induction, which showed that the cells had normal mammary cell function.(4) By Real Time PCR, we found the expression of anti-oncogene p53, p16, p14 and p21 decreased, which suggested that expression of anti-oncogene could be induced to be decreased by hTERT, and made the bovine mammary cell immortalization.(5) The bovine mammary stem cells were isolated by suspension culture. The bovine mammary stem cells expressedβ1-integrin,α6-integrin, Oct4, Nanog and Sox2 by immunochemistry, RT-PCR and western blot. In vitro the mammary stem cells could differentiate into mammary epithelial-like cells, myoepithelium-like cells and ductal-alveolar structure.(6) The BMECs and the BMECs transfected hTERT (hTERT-BMECs) were used as donor cells for comparing the development of nuclear transferred (NT) embryoes, we founded that telomerase could not improve the NT-embryoes development. And which domenstrated that the hTERT-BMECs we got could be used for NT.