胞壁酰二肽对体外奶牛乳腺上皮细胞生长及胞内NOD2 mRNA表达的影响  

Growth and Expression of NOD2 mRNA in Bovine Mammary Epithelial Cells Treated with Different Concentrations of MDP in Vitro

作　　者：徐丹丹[1] 王建发[1] 张旭[1] 刘东宇[1] 许小楠[1] 王乐[1] 陈嘉[1] 单旭菲 王晓雅[1] 武瑞[1] 杨彬[1] 

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319

出　　处：《中国农业科学》2017年第3期574-581,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31472249;31402157);黑龙江八一农垦大学研究生创新项目(YJSCX2015-Y23)

摘　　要：【目的】奶牛乳房炎是奶牛养殖业中最为常见,同时也是带来经济损失最为严重的疾病之一,其主要病因是细菌感染。奶牛乳腺的固有免疫是抵抗病原菌入侵的第一道防线。NOD2是机体固有免疫模式识别受体核苷酸结合寡聚域(nucleotide-binding oligomerization domain,NOD)蛋白家族中的重要一员,通过识别其特异性配体胞壁酰二肽(muramyl dipeptide,MDP)——一种广泛存在于革兰氏阳性菌和革兰氏阴性菌细胞壁中的成分,而参与抵抗多种病原菌入侵。奶牛乳腺上皮细胞(bovine mammary epithelial cells,BMEC)除泌乳以外,还是奶牛乳腺的免疫屏障。本试验欲探究MDP对BMEC体外生长状态及胞内NOD2表达量的影响。【方法】选取健康泌乳中期荷斯坦奶牛乳腺腺泡为组织原材料,采用胶原酶Ⅰ消化法结合梯度浓度胰蛋白酶纯化法分离BMEC;使用上皮细胞特异性表达的角蛋白-18(cytokeratin-18,CK-18)及成纤维细胞特异性表达的波形蛋白(Vimentin)抗体,通过免疫荧光技术对纯化后获得的细胞进行鉴定;将BMEC设为6个处理组,分别添加浓度为0(空白对照组)、1、5、10、15及20μg·mL^(-1)的MDP,24 h后显微镜下观察细胞状态,同时提取细胞RNA并反转录为cDNA,采用实时荧光定量PCR方法检测BMEC中NOD2的表达量。【结果】(1)胶原酶Ⅰ消化法结合梯度浓度胰蛋白酶纯化法分离得到的细胞CK-18免疫荧光结果为阳性,而Vimentin反应为阴性;细胞生长状态良好。(2)空白对照组、1、5及10μg·mL^(-1) MDP刺激组的BMEC生长状态良好,无任何肉眼可见变化;15μg·mL^(-1) MDP刺激组可见少量的BMEC脱落;而20μg·mL^(-1)MDP刺激组可见大量BMEC脱落,漂浮,且即使仍贴壁的细胞,其形态也发生了变化。(3)与空白对照组相比,各刺激组细胞NOD2 mRNA的表达量与MDP的刺激浓度呈正相关,即刺激时间为24 h时,随着MDP浓度的增加,BMEC中NOD2受体mRNA的表达量逐渐增加(P<0.05)。【结论[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 of NOD2 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.

关 键 词：奶牛乳腺上皮细胞 MDP NOD2 

分 类 号：S858.23[农业科学—临床兽医学]