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目的确定CFSE标记小鼠淋巴细胞的条件,建立简单快速、稳定可行的淋巴细胞体内示踪方法。方法利用流式细胞术分别检测不同浓度CFSE、不同悬浮溶液(1640培养基、PBS溶液)对小鼠淋巴细胞标记效果的影响,确定合适的标记条件;输注同品系CFSE标记的小鼠淋巴细胞,于不同时间点检测受鼠主要脏器内CFSE+细胞在淋巴细胞中的比例,并确定供鼠细胞的注射剂量;按照合适的注射剂量分别在注射后不同时间点测定供鼠细胞在受鼠体内的分布,考察本方法的稳定性及阳性信号的持续时间。结果 PBS组CFSE+细胞平均荧光强度分别为596、793、1123、1596、1737,明显高于1640培养基组(P<0.05);1640培养基组CFSE+细胞死亡率分别为8.7%、8.4%、8.6%、9.0%(P>0.05),而PBS组CFSE+细胞死亡率随着CFSE终浓度的上升而增加(P<0.05);按照不同剂量输注同品系淋巴细胞24h后,在受鼠各组织中检测到CFSE+细胞;每只小鼠尾静脉注射5×106个CFSE+淋巴细胞后,到d63仍可在组织中检测到阳性信号细胞。结论以PBS作为孵育液、CFSE终浓度选择10μmol/L对小鼠淋巴细胞标记、注射剂量为细胞5×106个/只,可使CFSE+细胞信号在同系小鼠体内持续2个月。
Objective To determine the condition of CFSE-labeled mouse lymphocytes and establish a simple, rapid and stable lymphocyte tracking method in vivo. Methods The effects of different concentrations of CFSE and different suspension solutions (1640 medium, PBS solution) on the labeling effect of lymphocytes in mice were detected by flow cytometry, and the appropriate labeling conditions were determined. The CFSE-labeled mouse lymphocytes , The ratio of CFSE + cells in the lymphocytes in the major organs of the rats was detected at different time points and the injection dose for the murine cells was determined. According to the appropriate injection dose, the donor cells were tested at different time points after injection Of the distribution, study the stability of the method and the duration of the positive signal. Results The average fluorescence intensity of CFSE + cells in PBS group was 596,793,1123,1596,1737, which was significantly higher than that in 1640 medium group (P <0.05). The CFSE + cell death rate in 1640 medium group was 8.7%, 8.4%, 8.6% %, 9.0% (P> 0.05). The CFSE + cell death rate increased with the increase of CFSE final concentration in PBS group (P <0.05) CFSE + cells were detected. After the mice were injected with 5 × 106 CFSE + lymphocytes through the tail vein, positive cells could still be detected in the tissues by d63. Conclusion With PBS as the incubation solution, the final concentration of CFSE was 10μmol / L, and the lymphocytes were labeled with 5 × 106 cells / CF CFSE + cells in syngeneic mice for 2 months.