The Effect Of Heat Stress On The HSP70mRNA Expression Of Cultured Dairy Cattle Mammary Epithelial Cell

Select healthy lactating mammary gland of Holstein cows, put it into the D-hank's solution with double-antibody,take back to the laboratory. we obtained the mammary epithelial cells by trypsin digestion, and cultured it in the six orifice which covered with rat tail collagen. The inoculation density was 1×106 / mL. Culture medium composed of DMEM/F12, fetal bovine serum, insulin, transferrin, epidermal growth factor, glutamine, double anti-so. Collected the cell medium after cultureed 48h to identify of whether the cells were mammary epithelial cells. After the cells adhered 48h, divided into two groups and heat treatment ,respectively.The first group dealed with a single high-temperature processing: put the 37℃normal cultured mammary epithelial cells in 38℃, 39℃, 40℃, 41℃water bath pot, immediately replace it into the 37℃, 95% humidity, 5% CO2 environment for cultivation after heated 1 hour . At the resumption of cultured 0h, 6h, 12h, 24h to collect cell samples, -70℃cryopreservation. The second group dealed with continuous heat treatment , every 24h do a high temperature for 1h, 38℃, 39℃, 40℃, 41℃water bath, the same way as before, after 24h each treatment to collect cell samples, continuous collected 3 days, -70℃cryopreservation. Extraction of total cellular RNA, reverse transcription, RT-PCR, gelelectrophoresis, recovery, and pGEM-T vector, transformed, shaking bacteria, plasmid was extracted, restriction enzyme digestion, sequencing, dilution steps of HSP70 and GAPDH standard plasmid preparation and Real time RT-PCR standard curve, quantitative determination the expression of these HSP70mRNA of the samples treated with different heat stress.By Western-blotting detection, the primary cultured cells were mammary epithelial cells. After the heat stress treatment, the expression of HSP70mRNA was positively correlated with temperature. And after a single heat stress treatment, the highest expression of HSP70 was at he resumption of cultured 6h ; the different expression of HSP70 between the 12h and 24h group groups was not significant (P> 0.05), both groups were significantly different with the 0h group (P <0.05) , and 6h extremely significant difference (P <0.01).Illustrated that cells returned to normal conditions after a sigle heat stess, the influence by heat stress gradually disappeared. Continuous heat treatment method showed that the expression of HSP70 mRNA of the 48h group was higher than the 24h group (P <0.05), but the 72h group HSP70 expression decreased . It shows that cells by a continuous heat stress in the process were damaged larger. In addition, after the continuous heat stress, the cells decreased HSP70mRNA expression, indicating cell may produce a heat tolerance. Effects of heat stress on dairy cows is extremely serious, serious heat stress can decrease milk production yield 80%, caused huge losses to cow breeding areas. This study was designed to the principle of heat stress on dairy cows in the field of molecular exploration,to study the heat stress mechanism of diary cow epithelial cells and to provide a theoretical basis for the nutritional regulation of heat stress.Not only were primary cultured bovine mammary epithelial cell in the cell type, cell characteristics, cell structure and cell function similar to the primary cell ,but also in the process to eliminate other influence factors, provides quick and convenience for our study.