流式细胞术检测单核巨噬细胞吞噬荧光素标记结核分枝杆菌的方法学探讨

目的：建立流式细胞术检测单核巨噬细胞吞噬荧光素标记结核分枝杆菌（Mycobacterium tuberculosis,Mtb）的方法。方法：用不同浓度荧光素（FITC）标记Mtb,流式细胞术检测FITC对Mtb的标记率。健康人外周全血与FITC标记Mtb（FITC-Mtb）于37℃共孵育10~120 min,溶解红细胞,洗涤后加台盼蓝淬灭未吞噬的FITC-Mtb的荧光,流式细胞术检测FITC阳性单核细胞的数值,计算单核细胞对Mtb的吞噬率。FITC-Mtb与佛波醇酯（PMA）激活分化的THP-1细胞以10∶1的比例共孵育1~6 h,或以1∶1~100∶1的比例孵育1 h和2 h,用同样的方法检测吞噬率。结果：FITC（50μg/ml）与Mtb作用2 h的标记率达92%。人单核细胞对FITC-Mtb的吞噬率从10 min的34.68%增加至120 min的79.90%。THP-1细胞对FITC-Mtb的吞噬率从1 h的30%增加至6 h的81%。FITC-Mtb与THP-1细胞以100∶1比例孵育1 h,吞噬率达平台（81%）;以50∶1的比例孵育2 h,吞噬率达平台（82%）。未加台盼蓝淬灭时,单核细胞在10 min和120 min对FITC-Mtb的吞噬率与加台盼蓝淬灭相比,分别增加29%和6%;而未加台盼蓝淬灭时,THP-1细胞在1 h和6 h对FITC-Mtb的吞噬率与加台盼蓝淬灭相比分别增加1%和7%。结论：流式细胞术检测人单核巨噬细胞吞噬FITC标记Mtb是测定单核巨噬细胞对Mtb吞噬活性的简便、快速和重复性好的方法。

A method for detection of monocyte/macrophage phagocytosis of fluorescent labeled Mycobacterium tuberculosis by flow cytometry

Objective： To establish a method for detection of monocyte/macrophage phagocytosis of fluorescent labeled Mycobacterium tuberculosis（Mtb） by flow cytometry. Methods： Mtb was labeled with fluorescein isothiocyanate （FITC） at different concentrations. The efficient labeled rate was detected by flow cytometry. Whole blood taken from healthy subjects were incubated with FITC labeled Mtb at 37 ℃ from 10 min to 120 min, followed by lysing red blood cells, washings, and then quenched the fluorescein of Mtb that not phagocytosed with trypan blue. The amount of monocytes with positive FITC were measured as phagocytosis rate by flow cytometry, FITC labeled Mtb and phorbol myristrate-differentiated THP-1 cells as macrophages were incubated from 1 h to 6 h with the ratio of 10：1 ,or incubated for 1 h and 2 h at the ratios from 1：1 to 100： 1, and the phagocytosis rates were detected by flow cytometry as same method above. Results：The efficient labeled rate for Mtb was 92% when incubated with FITC at the concentration of 50 μg/ml. The percentages of monocyte phagocytosis of Mtb were increased from 34.68% to 79.90% in coculture time from 10 min to 120 rain. The percentages of THP-1 macrophage phagocytosis of Mtb were increased from 30% to 81% for 1 h and 6 h of cocuhure. The THP-1 cell phagocytosis of Mtb were reached platform when FITC labeled Mtb incubated with THP-1 cells with at the ratio of 100：1 for 1 h（81% ） ,or at the ratio of50：1 for 2 h （82%）. After quenched with trypan blue, the percentages of monocyte phagocytosis of Mtb decreased by 29% in 10 min and by 6% in 120 min,whereas the percentages of THP-1 cell phagocytosis of Mtb decreased by 1% in 1 h and 7% in 6 h. Conclusions：The application of flow cytometry to measure monocyte/macrophage phagocytosis of FITC labeled Mtb is simple,rapid,and reproducible method.