Rapid microassays of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro

Simple, rapid microassays for simultaneous measurement of phagocytosis, bacterial killing, superoxide and hydrogen peroxide production by human neutrophils in vitro are described. All assays employ 96-well flat bottom tissue culture plates which were incubated on a microtitre plate shaker at 37 degrees C. The separate evaluation of ingestion and intracellular killing of E. coli and S. aureus was based on the incorporation of [3H]uridine into viable extracellular bacteria. There was good correlation between plate counts of viable bacteria and amount of radiolabel incorporation. Phagocytosis and killing can be measured in a maximum of 100 microliter reaction mixture, requiring only 2.5 X 10(5) neutrophils per test and the assay is complete within 60 min. Assay of superoxide production by stimulated neutrophils was based on superoxide-dependent reduction of ferricytochrome c as measured spectrophotometrically at 550 nm in wells of tissue culture plates containing 150 microliter of reaction mixture. The assay requires only 1.25 X 10(5) neutrophils per test and is complete within 50 min. Quantitation of hydrogen peroxide was based on horseradish peroxidase-dependent oxidation of phenol red. The technique is as for superoxide detection except that the reaction must be terminated by the addition of 1 M NaOH at the desired time intervals. None of the assays require sampling during the incubation period. The principal advantages of the described techniques are increased simplicity and speed, requirement of low numbers of neutrophils and applicability to analysis of large number of samples in parallel.     

银杏叶提取物抗鼠肾缺血再灌注损伤及对P选择素表达的影响

目的 :探讨银杏叶提取物保护大鼠肾脏缺血再灌注损伤作用与机理。方法 :SD大鼠随机分为假手术组、缺血再灌注组、银杏叶提取物组。通过HE染色观察肾组织损伤和中性粒细胞浸润 ,免疫组织化学方法观察P选择素的表达情况。结果 :与假手术组相比 ,缺血再灌注组肾小管上皮细胞呈明显的缺血性改变 ,P选择素表达明显增强 (P <0 .0 1) ,肾小球内中性粒细胞增加明显 (P <0 .0 0 1) ;银杏叶提取物组比缺血再灌注组肾小管上皮细胞缺血性改变减轻 ,P选择素表达减少 (P <0 .0 1) ,肾小球内中性粒细胞较缺血再灌注组减少 (P <0 .0 0 1)。结论 :银杏叶提取物减轻大鼠肾缺血再灌注损伤 ,减少P选择素表达及中性粒细胞浸润     

毒素清对肺炎双球菌肺炎老龄大鼠肺脏免疫功能的影响

目的 :从肺脏免疫功能探讨毒素清治疗老年肺炎的机制。方法 :复制肺炎双球菌肺炎老龄大鼠模型 ,分为对照组 ,模型组 ,毒素清高、低剂量组 ,头孢氨苄组 ;观察各组肺脏组织病理改变和肺泡灌洗液免疫球蛋白、纤维连接蛋白 (FN )和中性粒细胞计数。结果 :肺炎老龄大鼠肺组织损伤明显 ,与对照组比较 ,模型组分泌型Ig A (s Ig A)和 FN降低 ,Ig A和中性粒细胞增高 ;与模型组比较 ,用药各组 Ig A含量降低 ,毒素清高剂量组s Ig A、FN含量升高和中性粒细胞降低。结论 :肺脏中性粒细胞和免疫球蛋白含量增多及 s Ig A、FN含量减少与老年肺炎的发生发展有着密切关系 ;毒素清对肺炎的作用机制与其降低中性粒细胞数量和提高 s Ig A、FN含量等有关     

重组白细胞抑制因子和水蛭肽嵌合蛋白质量标准研究

目的:建立重组白细胞抑制因子和水蛭肽嵌合蛋白(TNHH)的质控方法和质量标准.方法:采用抗凝血酶活性试验和抗白细胞黏附试验测定活性;通过胰蛋白酶消化分析该蛋白还原型肽图;利用RP-HPLC法和SEC-HPLC法分别测定有关物质和高分子蛋白质含量;其余检测项目均按<中华人民共和国药典)2005版三部相关规定进行.结果:用建立的方法分别对一批TNHH原液和成品进行了检定,各项指标均符合<入用重组DNA制品质量控制技术指导原则>和现版<中华人民共和国药典>的要求.结论:建立的质控方法可有效地控制TNHH产品质量,并可用于TNHH原液及成品的常规检定.