关键词: ：奶牛乳腺上皮细胞, MDP, NOD2

Abstract: 【Objective】 Dairy cow mastitis is one of the most common diseases causing serious economic losses in dairy-farming industry. Bacterial infection is the main cause of mastitis. Innate immunity is the first line of defense against the invasion of pathogenic bacteria in mammary gland. NOD2 is an important member of the innate immune pattern recognition receptor of nucleotide-binding oligomerization domain (NOD) family, which defenses against various microbial invasions by recognizing its specific ligand-muramyl dipeptide (MDP), a component widely existing in gram positive bacteria and gram negative bacteria cell wall. Bovine mammary epithelial cell (BMEC) is the immune barrier of dairy cow mammary gland other than secreting milk. Here, the effect of MDP on the in vitro growth state of BMEC and the expression ofNOD2 in the BMEC was explored in this experiment.【Method】 Mammary gland tissue of healthy and lactating Holstein cows was chosen as raw materials. Collagenase digestion method combined with concentration gradient of trypsin was used to separate BMEC. Cytokeratin-18 specific expression in epithelial cells and vimentin specific expression in fibroblasts were used to identify the obtained cells by immunofluorescent techniques. BMEC was set to 6 treatment groups, including MDP stimulating concentrations of 0 (control group), 1, 5, 10, 15 and 20 μg·mL^-1. Twenty-four hours of poststimulation, BMEC status were observed under a microscope, meanwhile total RNA was extracted from BMEC and reverse transcribed to cDNA. Real time fluorescent quantitative PCR method was used to detect the expression of NOD2 in BMEC.【Result】Those cells separated by collagenase digestion method combined with concentration gradient of trypsin, immunofluorescence results of CK-18 reaction was positive and vimentin reaction was negative. All cells were in a good growth condition. In the control group and in the groups of MDP stimulating concentration at 1, 5 and 10 μg·mL^-1, BMEC grew well without any visible abnormalities. There was a small amount of BMEC detached from bottom in the group of MDP stimulating concentration at 15 μg·mL^-1. However, the group of MDP stimulating concentration at 20 μg·mL^-1 showed a large number of BMEC detached and floated from bottom. Even though those BMEC were still attached to the bottom, their morphology had already changed. Compared with the control group, the expression of NOD2 mRNA in BMEC was positively correlated with the stimulating concentrations of MDP. In other words, 24 h of poststimulation, the expression of NOD2 mRNA in BMEC gradually increased along with the stimulating concentrations of MDP.【Conclusion】High purity BMEC was successfully obtained. The obtained cells grew well and could be used in the following experiments. Although the expression of NOD2 mRNA was positively correlated with the stimulating concentrations of MDP, the stimulating concentrations of MDP in vitro culture BMEC should be controlled below 10 μg·mL^-1 in order to maintain the normal growth condition. These results suggested that BMEC could participate in the immune defense response of bovine mammary gland through the NOD2 receptor pathway. But this defense capability was influenced by the number of bacteria or the intensity of bacterial virulence. In a certain number or virulence of bacteria, the immune defense response of bovine mammary gland was enhanced along with the increasing number of bacteria or the enhancement of virulence to eliminate intramammary pathogens. While the number or virulence of bacteria exceeded to a certain range, bovine mammary gland tissue would be seriously damaged, so the immune defense barrier would be collapsed. Under this condition, the local bovine mammary gland or even all over the body would present obvious clinical symptoms.

Key words: bovine mammary epithelial cell, MDP, NOD2