The Study On Gene Expression Of Neutrophils Apoptosis Induced By Staphylococcus Aureus In Cows

Bovine mastitis is the largest affecting disease in dairy industry worldwide. Staphylococcus aureus, in a number of pathogens that can cause mastitis, is the most harmful. Due to the overuse of antibiotics and unsound of vaccines and other biological products, the prevention and treatment of mastitis becomes more difficult. Yet, the fundamental reason lies in the unclear of the infection and injury mechanism of staphylococcus aureus mastitis at the present stage, so it is urgent to explore the molecular mechanisms of staphylococcus aureus mastitis, either in the phase of occurrence, development or turnover.Neutrophil is the first line to prevent the bovine mammary from pathogen invasion. The apoptosis of neutrophils is essential for the control of the development and outcome in breast inflammation, and it play an important role in relieving unhealth inflammatory reaction in cows.In order to explore the molecular mechanism of apoptosis of neutrophils in bovine staphylococcus aureus mastitis, and find the regulation genes related to neutrophil apoptosis, we isolate neutrophils from the peripheral blood of cows in vitro and culture under the conditon of37℃,5%CO2. Then staphylococcus aureus, which is in the logarithmic growth phase, were added to the culture medium in a ratio of50:1. After co-culture of Oh,2h,4h,6h,8h and10h, neutrophils apoptosis was detected by flow cytometry using the method of FITC/PI double staining. Finally, the neutrophils apoptosis related genes were screening by genechips and the results were verifying by qPCR close behind. The experimental results obtained as follows:1. The survival of neutrophils cultured in vitroThe proportion of living cells declining with the prolonged incubation time. At Oh, it is100%; after cultured for10h and28h, the proportion of living cells reduced to80%and50%respectively.2. Apoptosis of neutrophils induced by staphylococcus aureusIn the induction time of Oh,2h,4h,6h,8h and10h, the percentage of apoptosis cells has significant differences between experimental group and control group, the difference values were3.31%,4.63%,6.77%,8.83%,8.12%and6.35%, and it has the biggest difference in the induction time of6h.3. The results of genechipsThere are four differentially expressed genes related to apoptosis. The up-regulated gene is Bcl-2with a multiple of2.4847. The down-regulated genes are IL1alpha, IL1beta and IL1-R1with multiples of0.2125,0.2062and0.3082.4. The results of qPCR validationThe expression trend of IL1alpha, IL1beta and IL1-R1in qPCR validation are consistent with that in genechips, but the results of Bcl-2are converse between the two methods, it performes down-regulated.The following conclusions can be obtained from the above results:1. Neutrophil in vitro cultural environment is capable of spontaneous apoptosis, and it has a shorter survival time.2. Staphylococcus aureus can induce apoptosis of neutrophil which is cultured in vitro, and changes in the rate of apoptosis within10h induction time are very significant.3. The main genes that regulate neutrophil apoptosis are proto oncogene gene Bcl-2, IL1and its receptor.4. The results of genechips are credible.