MTT法测定奶牛血液及乳中多形核白细胞的杀菌功能  

A MTT Colorimetric Assay for Determination of Bactericidal Activity of Bovine Polymorphonuclear Leukocytes

作　　者：母安雄[1] 胡松华[1] 

机构地区：[1]浙江大学动物科学学院,浙江杭州310029

出　　处：《中国兽医学报》2001年第2期141-144,共4页Chinese Journal of Veterinary Science

基　　金：浙江省自然科学基金资助项目! (3972 6 5 )

摘　　要：金黄色葡萄球菌和多形核白细胞于 37℃培养一定时间 ,用皂素破坏白细胞 ,然后加入 3-( 4 ,5-二甲基噻唑 -2 -YL) 2 ,5-二苯基 -四唑溴盐 ( MTT)溶液。未被白细胞杀死的金黄色葡萄球菌将 MTT转化成蓝紫色甲月赞沉淀。用二甲基亚砜溶解沉淀 ,并用酶标仪测定甲月赞相对含量 ,以此推算白细胞杀菌能力。结果表明 ,MTT法重复性好 ,一次可测定多个样品 ;与平板计数法比较 ,有较好的一致性。用该法测定奶牛血液和乳中白细胞的杀菌功能 ,结果牛乳中白细胞杀菌功能显著低于血液中的白细胞 。A colorimetric assay was used for quantitating bactericidal activity of bovine neutrophil against Staphylococcus aureus. The polymorphonuclear leukocytes were incubated with S. aureus, and then lysed with saponin. The tetrazolium compound, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), was added.. The viable bacteria reduced MTT to purple formazan, which was easily dissolved in DMSO and quantitated by measuring absorption in a ELISA-reader. The production of formazan was used to estimate the percentage of bacteria killed by neutrophils. The method had a high sensitivity and reproducibility as well as good correlation with the colony-counting method, but was easy to perform and could detect several samples once time. We used the approach to test the bactericidal activity of the polymorphonuclear leukocytes (PMN) isolated from bovine blood and milk. The bactericidal activity of PMN was much lower than that of blood PMN. This may be responsible for the high suceptibility of the bovine mammary gland to bacterial infection.

关 键 词：多形核白细胞 奶牛 杀菌功能 天噬细胞 MTT法 免疫 

分 类 号：S852.42[农业科学—基础兽医学] S858.232.4[农业科学—兽医学]