流式细胞术检测大鼠肺泡巨噬细胞吞噬功能方法探讨

A flow cytometric assay to detect phagocytic function of alveolar macrophage

目的:比较不同实验条件对检测肺泡巨噬细胞吞噬功能的影响,为流式细胞术检测肺泡巨噬细胞吞噬功能筛选快速、灵敏、稳定的实验方法。方法:支气管肺泡灌洗获取肺泡巨噬细胞,将肺泡巨噬细胞分别与转红色荧光蛋白基因质粒细菌〔E.coliDH5α(pJZ402rfp)〕及未转质粒细菌(E.coliDH5α)孵育,并选择〔E.coliDH5α(pJZ402rfp)〕与肺泡巨噬细胞按不同的细菌与细胞比例(50:1、100:1、200:1)、孵育时间(30min、60min、120min、180min)及培养溶液(PBS、Hanks、无血清1640)进行孵育,流式细胞仪检测肺泡巨噬细胞的吞噬功能。结果:E.coliDH5α(pJZ402rfp)具有稳定的红色荧光,用E.coliDH5α(pJZ402rfp)检测肺泡巨噬细胞的吞噬功能比用E.coliDH5α获得更多的参数。肺泡巨噬细胞的吞噬率随着细菌与细胞比例的增大而增加,而吞噬指数在细菌与细胞比例为100:1时为最高(P<0.05);肺泡巨噬细胞的吞噬率从30分钟到120分钟变化不大,但到180分钟吞噬率有明星的降低(P<0.05),吞噬指数则在60分钟时最高,有显著差异(P<0.05);Hanks液组肺泡巨噬细胞的吞噬率及吞噬指数显著高于PBS组及1640组(P<0.05)。结论:在室温(260C)以肺泡巨噬细胞与E.coliDH5α(pJZ402rfp)比为1:100的Hanks液中孵育1小时,是流式细胞术检测肺泡巨噬细胞吞噬功能的快速、灵敏、稳定的实验方法。

ObjectiveThe purpose of this study was to establish a flow cytometric assay on the phagocytic function in rat alveo- lar macrophage （AM） Methods AM collected by bronchoalveolar lavage from rats phagocytes E.coliDH5a（pJZ402rfp） in different condition. The percentage of positive AM with associated bacteria was determined as phagocytic rate, and phagocytic index was de- tected as ration of fluorencence index （FI） of positive AM to FI of control AM, which were analyzed by flow cytometry. Results The red fluorescent protein of E.coliDH5ct（pJZ402rfp） is bright and stabilized. There are more parameters obtained from AM chal- lenged with E.coliDH5u（pJZ402rfp） than E.coliDH5ct. Phagocytic rate in AM increased with the ration of bacteria to AM. Howev- er, the highest phagocytic index was found in the ratio of 100：1 （P〈0.05） Phagocytic rate decreased markedly at 180min, com- pared with 30, 60, and 120 min. While the phagocytic index significantly increased at 60min （P〈0.05） Compared with PBS group and 1640 group, phagocytic rate and phagocytic index markedly increased in Hanks group （P〈0.05） Conclusions AM phago- cytes E.coliDH5a（pJZ402rfp） with the ration of 1：100 in the Hanks for 60 min at RT. We have developed a flow eytometric assay to detect ohagocvtic rate and nha^ocvtic index in AM.