Periodicity Rule And Mechanism Of Apoptosis In Dairy Cow Mammary Epithelial Cells

Apoptosis is programmed cell death process, is an essential physiological process that plays a critical role in development and tissue homeostasis. The breast is a renewable tissue, which to go through the process of cell proliferation and apoptosis in each pregnancy cycle, in particular, after weaning, the breast gets into a degradation process, along with a large number of mammary epithelial cell apoptosis. Because apoptosis is a necessary stage that breast realize its function, so it can be better to understand the change rule of breast development structure, find the biological control methods to reasonablely prolong lactation and increase milk yield through systematically research periodic rule and its mechanism of apoptosis in mammary gland development. Effective regulation of the function of cows at the genetic level, it can improve dairy cow yield, improve the conversion rate of nutrients and ameliorate milk quality. It calso can improve the economic efficiency of dairy fanning and solve some major issues facing the field of dairy industry in China.In this study, Holstein dairy cows were used as the experimental materials, TUNEL was used to detect the apoptosis of mammary cell, mammary gland tissues of different developmental stages were sliced in frozen, to detect the apoptosis single in dairy cow mammary gland tissue and to determine the apoptosis rule at different development stages in dairy cow mammary gland tissue. Applying fluorescence quantitative RT-PCR and western blotting techniques, the expression of a series apoptosis genes and proteins at different development stages in dairy cow mammary gland tissue were detected, including Bax (apoptotic protein), TGF-β1, P53gene, and to verify expression of important signal apoptosis-related protein, including caspase3(downstream), Bax (upstream), TNF-R1(upstream)TUNEL results showed that, in virgin, mammary gland developed so stablely and most breast tissue was the fat pad structure, in a period of isometric growth, simultaneously the extent of apoptosis was relatively low. In pregnancy, mammary gland developed rapid, with a large number of alveolus and apoptosis was increased. In initial pregnancy2m, under the action of the pipuitary, ovarian and placental secretion of many hormones, the ductal system and alveolus developed rapidly, appeared a secondary apoptosis peak. In lactation, the structure of mammary gland was stable, little newborn and degradation of structural changes and apoptotic signal was almost undetectable. Late lactation until involution, gland alveolus began to open and gradually transited to involution, alveolar epitheial cells gradually began to undergo apoptosis, the apoptotic signal increased than in early lactation. In involution3d, the apoptosis was highest and reached the peak, green positive signal was almost full of screen, and then decreased. In involution30d, the mammary gland was recovered to the virgin status, the amount of apoptosis was also reduced, to start the next circulation. Real-time PCR and western blotting results showed that apoptosis genes expressed in the different development periods of dairy cow mammary gland, but expresstion were not consistent. The result of fluorescence quantitative RT-PCR showed that P53was expressed in every periods, it was greatest in late virgin to pregnancy and significantly up-regulated in the virgin (P<0.05). Expression of transforming growth factor β(Tgf-β) mRNA was greatest in late virgin, significantly higher than other periods (P<0.05). Fas was significantly up-regulated in the late lactation (P<0.05). Tnf-r1was experssed in every period and showed dynamic state changes. BCL2-associated X protein (Box) gene expression was greatest during the early pregnancy and late lactation and coincided with high cell turnover. The downstream gene caspase-3was significantly up-regulated in the late involution (P<0.05). Western blotting analysis also showed the protein dynamic changes in expression levels. Based on TNF-R1(tumor necrosis factor), caspase-3, and caspase-8gene expression, no indication was found of a stage-dependent shift between the extrinsic and intrinsic pathways leading to apoptosis.