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目的:利用CFSE标记细胞,流式细胞术(FCM)检测法,解析超抗原SEB活化的耐受性CD8+ NKT细胞在体外增殖的情况。方法:利用CFSE标记新鲜分离的C57BL/J鼠脾细胞,分别与ConA和LPS共同培养3d,收集细胞进行荧光染色并用FCM解析细胞表面CD69分子的表达率和增殖能力。CFSE标记的鼠脾细胞与SEB共培养5d和10d后,荧光染色并用FCM解析细胞表面CD69的百分数和增殖能力。SEB活化的第10天细胞经CFSE标记后在IL-2的协同作用下继续培养10d,荧光染色,FCM解析这群细胞的增殖能力、活性分子CD69的表达率和NKT细胞亚群的变化情况。结果:ConA、LPS和SEB三者均可以刺激小鼠脾细胞增殖。ConA和LPS在3d内可以使细胞增殖3代,且CD69的表达率为74.19%和41.56%;SEB在5d和10d内分别可以使细胞增殖5代和7代,细胞表面CD69的表达率为32.09%和48.66%。SEB活化的10d细胞可以在IL-2的协同下继续传代培养10d,可以增殖7代;这群细胞中CD8+ NKT细胞亚群,由原始的0.36%增加到38.58%;细胞表面CD69分子由正常值的0.11%提高到83.74%。结论:超抗原SEB活化的CD8+ NKT细胞可以在体外进行增殖培养,且这些细胞是活性化的细胞。利用CFSE标记细胞,FCM可以检测耐受性CD8+ NKT细胞在体外的增殖水平。
OBJECTIVE: To analyze the proliferation of CD8 + NKT cells activated by superantigen SEB by using CFSE labeled cells and flow cytometry (FCM). Methods: CFSE-labeled freshly isolated C57BL / J mouse splenocytes were co-cultured with ConA and LPS respectively for 3 days. The cells were collected for fluorescence staining and the expression of CD69 on the cell surface was analyzed by FCM. CFSE-labeled mouse spleen cells were co-cultured with SEB for 5 days and 10 days. Fluorescent staining and FCM were used to analyze the percentage of CD69 and proliferative capacity on the surface of the cells. The CFSE-labeled cells on the 10th day after SEB activation were cultured for 10 days under the synergistic effect of IL-2. Fluorescence staining and FCM were used to analyze the proliferation ability, the expression rate of CD69 and NKT cell subsets. Results: ConA, LPS and SEB can stimulate mouse splenocyte proliferation. ConA and LPS could proliferate for 3 generations in 3 days, and the expression rates of CD69 were 74.19% and 41.56%. SEB could proliferate 5 and 7 generations respectively on 5d and 10d, and the expression rate of CD69 on cell surface was 32.09 % And 48.66%. SEB-activated 10d cells could continue to subculture for 10 days in synergy with IL-2, and could proliferate for 7 generations. The population of CD8 + NKT cells in this group increased from 0.36% to 38.58%; the cell surface CD69 molecule was increased from normal 0.11% to 83.74%. CONCLUSIONS: Superantigen SEB-activated CD8 + NKT cells can be cultured in vitro in vitro and these cells are activated. Using CFSE labeled cells, FCM can detect the level of proliferation of tolerogenic CD8 + NKT cells in vitro.