创伤弧菌溶细胞素对小鼠肝脏CD4＋T细胞线粒体数量及CD62L表达的影响

Effects of Vibrio vuinificus cytolysin on the numbers of mitochondria and the expression of CD62L in murine liver CD4＋ T cells

目的研究重组创伤弧菌溶细胞素（rVvhA）对小鼠肝脏CD4＋T细胞的细胞毒活性及其对线粒体数量和CD62L表达中的作用，了解其对肝脏不同类型CD4＋T细胞存活率的影响。方法利用大肠埃希菌表达系统重组表达创伤弧菌溶细胞素，体外作用C57BL／6小鼠的肝脏单个核细胞，采用流式细胞术结合CD4、CD44、CD62L、Live／Dead、MitoTrackerRed和JC-1等荧光抗体（或探针）检测分析rVvhA对肝脏CD4＋T细胞及其亚群的存活率、线粒体数量和线粒体膜电位的影响。结果rVvhA体外作用6h后，小鼠肝脏CIM＋T细胞的存活率下降，rVvhA对CD4＋初始T细胞和效应性T细胞均有杀伤作用，且CD4＋T细胞CD62L的表达明显下调，此外，CD4＋T细胞的线粒体数量减少，线粒体膜电位显著下降。结论重组创伤弧菌溶细胞素对CD4＋T细胞的细胞毒活性可能与CD62L表达、抑制线粒体数量和膜电位有关。

Objective To understand how Vibrio vulnificus hemolysin （VvhA） affects the viability of routine liver CIM＋ T cells as well as its effects on the numbers of mitochondria and the expression of CD62L. Methods The primary murine liver monocytes （MNs） were isolated from C57BL/6 mice and then treated with recombinant VvhA （rVvhA） for 6 hours in vitro. The viability of routine liver CD4＋T cells and the expression of CD62L were measured by staining with anti-mouse CD4, CD8, CD44, CD62L and cell via- bility fluorescent dye or fluorescent antibody. Moreover, the cells were simply incubated with MitoTracker or JC-I probes to label mitochondria and mitochondrial membrane potential, which were further analyzed by using flow cytometry analysis. Results With the increase in the doses of rVvhA, the viability of routine liv- er CD4＋T cells was decreased from 81.5% to 15.8%. The expression of CD62L on the surface of murine liver CD4＋T cells was dramatically decreased. Both the murine liver nafve and effector CD4＋T cells were sensitive to the cytotoxicity of rVvhA. Moreover, treating routine liver CD4＋T ceils with rVvhA resuhed in significantly decreased numbers of mitochondria and lower mitochondrial membrane potential. Conclusion The cytotoxicity of rVvhA to murine liver CD4＋T cells might be achieved through inhibiting the expression of CD62L, decreasing the numbers of mitochondria and lowering mitochondrial membrane potential.