The Expression Of Primary Factors And Apoptosis In Bovine Mammary Epithelial Cells Infected S. Aureus,E. Coli

The cow mastitis is one of the major diseases which can affect dairy industry,which not only damages the health of dairy cows,but also leads to a decline in the quality of dairy products.The pathogenic microorganisms are the primary factors that induce the development of mastitis.Many pathogenic microorganisms can induce apoptosis and influence the occurrence and development of the disease.The study aimed at exploring the effects of S.aureus and E.coli infecting bovine mammary epithelial cells(BMECs)on cytokines and apoptosis expression.In this experiment which was performed by improving the two enzymes digestion method,BMECs were successfully cultured in vitro and that had been identificated by immunocytochemical.The 5th generation BMECs after purification were selected and added with different concentrations of S.aureus or E.coli,and the formula was used to calculate the infection volume as 0.6 mL to obtain the optimal infection concentration.We calculate through the formula best quantity 0.6 mL infected cells.The cells were infected by S.aureus or E.coli at an optimal concentration of 100:1 viable cell ratio(MOI),and sampled at 0,3,6,9 and 12 hours after infection.Real-time fluorescence quantitative PCR was used to detect the expression of IL-1β,IL1RⅠand TNF-αand Tunel was used to detect the apoptosis of S.aureus or E.coli infected cells.The results showed as follows:(1)BMECs were successfully cultured in vitro and the best method of cryopreservation was determined.And detected the cells by immunocytochemistry,the BMECs simultaneously expressed keratin 18 andβ-casein and did not express vimentin,verifying that the cultured cells were mammary epithelial cells.(2)After different concentrations of S.aureus treat on cells for 12 h,the relative mRNA expression levels of IL1RⅠ,IL-1β,and TNF-αwere highest at 10~2,10~4,and10~3 CFU/mL,respectively.After different concentrations of E.coli treat on cells for12 h,the relative mRNA expression levels of IL1RⅠ,IL-1β,and TNF-αwere highest at 10~1、10~3、10~4 CFU/mL,respectively.(3)The cells treated with S.aureus or E.coli with a concentration of 10~1-10~4CFU/mL respectively increased the apoptosis rate with the increase of the concentration of the bacteria.(4)The relative expression of IL1RⅠmRNA was highest when 10~2 CFU/mL S.aureus was added for 6 h.The relative expression of IL-1βmRNA was highest at 10~4CFU/mL S.aureus for 9 h,and the relative expression of TNF-αmRNA was highest at 10~3 CFU/mL S.aureus 12 h.The relative expression of IL1RⅠmRNA was highest when E.coli of 10~1 CFU/mL was added for 6 h,and the relative expression of IL-1βmRNA was highest when E.coli 10~3 CFU/mL was added for 9 h.The relative expression of TNF-αmRNA was highest at 10~4 CFU/mL E.coli for 12 h.In this study,it was found that the relative expression levels of IL-1β,TNF-α,and IL1RⅠgenes were different in S.aureus or E.coli infected cow mammary epithelial cells at different concentrations and at different time points.And found that different concentrations of S.aureus or E.coli infected cells,with the concentration of the bacteria increased the apoptosis rate increased.In order to further study the pathogenic mechanism of mastitis caused by bacterial infection,it provides a theoretical basis.