Mechanism Of Bovine Mammary Epithelial Cells Response To Hyperthermia

The aim of this paper is to reveal the mechanism of bovine mammary epithelial cells response to hyperthermia. Firstly, a bovine epithelial mammary cell culture system was established, a suitable hyperthermia treatment was determined, and the effects of hyperthermia on bovine mammary epithelial cell biology characteristics and its regulation mechanism were studied.1 Establishment of a bovine epithelial mammary cell line and identificationA simple method of trypsin /Collagenase I alternative digestion and iterative culture flask adherence to discard fibroblasts for bovine mammary cell culture was established in this study. By immunohistochemistry, flow cytometry, western blotting, electron microscopy analysis, the characteristics of bovine mammary cells were investigated in vitro. The results showed that the mammary cells were diploid epithelia with intact 30 pairs chromatins, which could secrete alpha-casein into the medium.2 The effect of hyperthermia on bovine mammary epithelial cell biological characteristicsBovine mammary epithelial cells were incubated at 40℃/41℃for 1 hours, and were measured by trypan blue dying, flow cytometry to study the effect of hyperthermia on cell cycle and cell death. The results showed that (1) After exposed to hyperthermia, the cell condensed chromatin like crescent on the nuclei verges, mitochondria occurred expansion and vacuolization, and apoptotic bodies appeared, which suggested that heat stress could induce mammary epithelia apoptosis. (2)Cell cycles were blocked at S phase and G₂/M phase, and apoptosis and necrosis rates were upregulated. (3) The mammary epithelial cells responded to hyperthermia developed thermotolerance.3 The function of mitochondria pathway in bovine mammary epithelial cell death induced by hyperthermia To investigate the influence of hyperthermia on bovine mammary epithelial cells, changes of cell proliferation, ultra-structural morphology, and mitochondria death pathway were observed after their exposure to 40℃for 1 h. The results demonstrated the cell death occurred in typical cascade, namely chromatin margination, mitochondrial swelling, mitochondrial membrane potential (△ψm) decreasing, ratio of Bax / Bcl-2 increasing, activities of caspase-3 dramatically increasing, and DNA fragmentation. These results indicate that hyperthermia induced bovine mammary cell apoptosis and necrosis through mitochondria triggering death pathway.4 HSP72 expression and a role of JNK kinase in the bovine mammary epithelial cells under heat stressBy the methods of RT-PCR, immunohistochemistry, flow cytometry and JNK activator/ inhibitor, HSP72 expression and a role of JNK in cell death pathway were studied. The results showed that (1) Hyperthermia induced bovine mammary epithelial cell HSP72 expression, which associated with thermotolerance. (2) JNK involved in mitochondria triggering death pathway.In conclusion, we revealed heat responses in bovine mammary epithelial cells. The results accomplished in this work show that hyperthermia inhibits cell proliferation, induces bovine mammary epithelial cell death by mitochondria triggering apoptosis / necrosis pathway, furthermore induces G₂/M arrest by DNA damage. On the other hand, hyperthermia induces HSP72 proteins expression which associated with thermotolerance.