流式细胞术检测胞内重组耻垢分枝杆菌的活力研究

目的建立一种快速检测胞内分枝杆菌活力的方法。方法将一定量培养至对数生长期的含pMV-eis的重组耻垢分枝杆菌感染U937巨噬细胞,以含空质粒的耻垢分枝杆菌为对照,吞噬作用2 h后洗去胞外细菌,再分别培养4、12、24和48 h后收集细胞并裂解之。获得的胞内细菌用FDA荧光染料染色后用流式细胞仪检测死亡率,并与平板菌落计数法进行比较。结果流式细胞仪检测出感染12 h后重组耻垢分枝杆菌胞内死亡率较对照组均有显著下降（P〈0.05）,流式细胞仪检测法与平板菌落计数法相比差异无统计学意义（P〉0.05）。结论流式细胞术与传统的平板计数法相比具有快速、敏感、方便的特点,可用于分枝杆菌活菌快速检测。

Study on intracellular growth test of recombinant Mycobacterium smegmatis by flow cytometry

Objective To detect and develop a rapid method for intracellular survival test of recombinant Mycobacterium smegmatis.Method U937 macrophages were infected with recombinant M.smegmatis containing plasmid pMV-eis,which were cultured to logarithm growth phase.M.smegmatis containing empty plasmid pMV261 was the control.After 2 h of phagocytosis period,extracellular bacteria was washed away,and then U937 macrophages were incubated at 37 ℃ in 5% CO2 for some time.At various times,the macrophages were lysed and intracellular bacteria were harvested.Then the bacteria were stained with FDA and detected by flow cytometry.The results of the apoptosis ratio of recombinant M.smegmatis in U937 cells testing by flow cytometry were compared to the ones by plate colony counting method.Result 12 h after infection,the mortality ratio of pMV-eis colonies in U937 cells significantly decreased relative to pMV261 colonies.The results obtained by flow cytometry and plate colony counting method were in excellent agreement.Conclusion Flow cytometry is a rapid,accurate and convenient method,and can be used for quick detection of survival mycobacteria in macrophages.