小鼠巨噬细胞系体外感染卡介苗的应答

Response of mouse macrophage cell line infected with Bacille Calmette-Guerin in vitro

【目的】探讨小鼠巨噬细胞系RAW264.7体外感染卡介苗的应答。【方法】体外感染RAW264.7细胞23h后,分析细胞形态和细胞表面共刺激分子的表达。然后去除培养上清中的卡介苗,继续培养不同时间,通过CFSE、annexin V/PI和Rh123标记,分析宿主细胞的应答。【结果】卡介苗感染23h后,细胞生长状态良好,细胞内能明显观察到吞噬泡中的BCG。细胞表面共刺激分子CD40、CD54、CD80、CD86、CD11b的表达明显升高,CD11c、I-Ad以及H-2Kd的表达变化不明显。CFSE标记卡介苗后,随着培养时间的延长,荧光强度逐渐减弱,但是4天后仍然明显地高于对照组。除去培养上清中的卡介苗后继续培养,含有卡介苗的细胞逐渐减少,继续培养60h后基本检测不到。另外,卡介苗感染不能诱导细胞凋亡,线粒体膜电位先升高后降低,5d后,基本上与对照组一致。【结论】通过以上分析,为卡介苗免疫机理的研究提供了重要数据。

[Objective]To illuminate the response of mouse macrophage cell line RAW264.7 after infected with Bacille Calmette-Guerin （BCG） in vitro. [Methods] We analyzed the morphology and expression of co-stimulatory molecules on the surface of RAW264.7 after exposure to BCG for 23 hours. Then during the different culture time after discard the BCG in the supernatant, we analyzed the response of RAW264.7 by flow cytometry using carboxyfluorescein diacetate, succinimidyl ester （CFDA-SE）, annexin V/PI and Rhodamine 123, respectively. [Results] We observed the phagosome containing BCG under transmission electronic microscope and RAW264.7 cells still grow well after incubated with BCG for 23 hours. We found that the expression level of co-stimulatory molecules, CD40, CD54, CD80, CD86 and CD11b, were elevated evidently, except CD11c, I-A^d and H-2K^d. The fluorescence intensity of BCG stained with CFDA-SE decreased with the prolongation of culture time, but it was still more higher than unstained control four days later. After removal of uninfected BCG, the percents of BCG-infected cell decreased and we didn’t detected BCG-infected cells evidently for 60 h culture. Furthermore, we also found that there was no obvious cell apoptosis for all the time, and the mitochondrial membrane potential of BCG-infected cells increased early and then decreased to the same level as the uninfected control. [Conclusion]These results would be helpful to elucidate the mechanism of BCG vaccination.