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目的探讨肺炎链球菌溶血素(pneumolysin,Ply)对人急性单核细胞白血病单核细胞株THP-1细胞的增殖抑制和诱导凋亡作用及机制。方法 MTT法检测Ply对THP-1细胞的增殖抑制。瑞氏染色观察Ply对人THP-1细胞形态的影响。AnnexinV-FITC/PI双标法检测细胞凋亡。琼脂糖凝胶电泳观察凋亡细胞DNA片段化现象。免疫组化和Western blot检测MAPK1/3蛋白表达的变化。结果将人THP-1细胞分别与0.05、0.1、0.5、1、2.5、5μg/ml Ply共同孵育后,在24、48、72、96 h用MTT法检测细胞增殖抑制率,发现Ply对人THP-1细胞具有明显的增殖抑制作用,并且呈剂量和时间依赖性,IC50(24 h)为1.31μg/ml;0.05μg/ml和0.1μg/ml Ply分别处理THP-1细胞12 h后,通过瑞氏染色法观察人THP-1细胞可见凋亡小体及核碎裂象等典型的凋亡形态学改变;用0.05μg/ml Ply和0.1μg/ml Ply作用THP-1细胞12 h后细胞早期凋亡率分别为11.83%和48.45%(P<0.05);提取凋亡THP-1细胞DNA,进行琼脂糖凝胶电泳可见典型的“梯状”条带;Ply作用于人THP-1细胞后,胞内MAPK1/3蛋白表达降低(P<0.05)。结论 Ply可诱导人THP-1细胞凋亡,此作用涉及MAPK1/3信号通路的参与。
Objective To investigate the effects of pneumolysin (Ply) on the proliferation and apoptosis of human acute monocytic leukemia cell line THP-1. Methods MTT assay was used to detect the inhibitory effect of Ply on proliferation of THP-1 cells. Wright’s staining observed Ply on human THP-1 cell morphology. AnnexinV-FITC / PI double-labeled method for detecting apoptosis. Sepharose DNA fragmentation was observed by agarose gel electrophoresis. The changes of MAPK1 / 3 protein expression were detected by immunohistochemistry and Western blot. Results After THP-1 cells were co-incubated with 0.05, 0.1, 0.5, 1, 2.5 and 5 μg / ml Ply, the cell proliferation inhibition rates were detected by MTT assay at 24, 48, -1 cells in a dose-and time-dependent manner, IC50 (24 h) was 1.31μg / ml; THP-1 cells were treated with 0.05μg / ml and 0.1μg / ml Ply for 12 hours respectively, Wright’s staining observed THP-1 cells showed apoptotic bodies and nuclear fragmentation and other typical apoptotic morphological changes; with Ply 0.05μg / ml and 0.1μg / ml Ply THP-1 cells after 12 h cells The apoptosis rates of THP-1 cells were 11.83% and 48.45%, respectively (P <0.05). The DNA of apoptotic THP-1 cells was extracted by agarose gel electrophoresis and typical “ladder” 1 cells, the intracellular MAPK1 / 3 protein expression decreased (P <0.05). Conclusion Ply can induce THP-1 cell apoptosis, and this effect involves the MAPK1 / 3 signaling pathway.