卡介菌多糖核酸对泌乳奶牛外周血PMN吞噬功能及活性氧释放的影响  

Effect of polysaccharide nucleic acid fraction of Bacillus Calmette Guerin on phagocytosis and generation of reactive oxygen species of peripheral blood neutrophil in lactating cow

作　　者：苗晋锋[1] 顾蓓蓓[2] 刘仪[1] 邹思湘[1] 

机构地区：[1]南京农业大学动物医学院,江苏南京210095 [2]泰州出入境检验检疫局,江苏泰州225300

出　　处：《中国兽医科学》2011年第8期811-816,共6页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31072104);南京农业大学青年创新基金项目(Y200917)

摘　　要：为探讨卡介菌多糖核酸(polysaccharide nucleic acid fraction of Bacillus Calmette Guerin,BCG-PSN)对泌乳奶牛外周血嗜中性粒细胞(neutrophil,PMN)吞噬功能及活性氧(reactive oxygen species,ROS)释放的影响,选取处于泌乳初期的健康奶牛,无菌采集颈静脉血,肝素抗凝。不同浓度的BCG-PSN(终浓度分别为0、2、5、10、40、80μg/mL)与全血共孵育2h,加入FITC标记的大肠杆菌悬液10μL,37℃20min后,放入冰水中终止吞噬反应;溶血素裂解红细胞,0.01mol/L、pH 7.2PBS洗涤2次,重悬后FL1通道检测PMN对大肠杆菌的吞噬率。不同浓度的BCG-PSN(终浓度分别为0、2、5、10、40、80μg/mL)与全血共孵育2h后,加入乙酸肉豆蔻佛波酯(phorbol myrisate acetate,PMA)刺激PMN,其中PMA刺激的终浓度为100ng/mL,同时每个浓度梯度设无PMA(等量PBS代替PMA)刺激对照;37℃作用15min后加入双氢罗丹明123(dihydrorhodamine 123,DHR 123),终浓度为5μmol/L,然后37℃孵育10min;溶血素裂解红细胞,0.01mol/L、pH 7.2PBS洗涤2次,多聚甲醛固定、重悬后流式细胞仪检测。试验结果显示,BCG-PSN可以降低PMA刺激引起的PMN的ROS的大量释放,且这种抑制呈剂量依赖性降低;而对PMN的吞噬功能没有明显的影响。结果说明BCG-PSN对PMN功能的影响不是通过直接影响其吞噬功能发挥作用;而降低炎症反应时PMN氧自由基的过度产生是BCG-PSN减轻机体炎症反应的原因之一。结果提示,BCG-PSN对奶牛乳腺炎的免疫生理调控有潜在的应用价值。The aim of this study was to evaluate the effect of polysaccharide nucleic acid fraction of Bacillus Calmette Guerin （BCG-PSN） on phagocytosis and generation of reactive oxygen species（ROS） of lactating cow peripheral blood neutrophil（PMN） in vitro. Peripheral blood of healthy lactation cow at early stages of lactation was collected from the jugular vein by venipuncture in evacuated tubes containing heparin as anticoagulant. FITC-labeled E. coli 10μL was added to each sample after a 2 h co-incubation with different concentrations of BCG-PSN（final concentration of 0,2,5,10,40 and 80μg/mL）. After further 20 min incubation at 37 ℃ ,phagocytosis was stopped by dipping into ice water. Then erythrocytes were lysed with lysing solution and the leucocytes were washed two times with PBS（0.01 mol/L,pH 7.2）. The cells were then resuspended in PBS and analyzed on a FACScan flow cytometer. FL1 green channel captured FITC fluorescence. Phorbol myrisate acetate（PMA, final concentration of 100 ng/mL） was used to stimulate PMN after a 2 h co-incubation with different concentrations of BCG-PSN（final concentration of 0,2,5,10, 40 and 80μg/mL）. Control tests of every group were set up with PBS. All tubes were incubated at 37 ℃ for 15 min followed with addition of working dihydrorhodamine 123 （DHR 123, final concentration of 5μmol/L） solution and incubated at 37 ℃ for 10 min. Then erythrocytes were lysed with lysing solution, washed two times with PBS（0.01 mol/L, pH 7.2） and stabilized by paraformaldehyde. The cells were re- suspended in PBS and analyzed on a FACScan flow cytometer. The results showed that BCG-PSN could significant reduced the generation of reactive oxygen species of PMN and had no effect on the phagocytosis of PMN. This suggests that BCG-PSN protects the mammary gland from inflammatory injury by attenuating the release of ROS but not by directly phagocytosis. BCG-PSN may be a potential candidate to prevent bovine mastitis.

关 键 词：卡介菌多糖核酸 PMN 吞噬 活性氧 泌乳奶牛 

分 类 号：S856.5[农业科学—临床兽医学]