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为探讨脐带血单核细胞向树突状细胞分化过程中EB病毒对凋亡的影响,无菌条件下采集足月顺产新生儿脐带血,体外分离纯化单核细胞,经rhIL-4和rhGM-CSF诱导使之向树突状细胞分化,病毒组在培养的第0天加入B95.8培养上清。Hoechst 33258染色观察细胞形态学变化,Annexin V-FITC/PI双标记流式细胞术检测凋亡率,Western blot检测X-连锁凋亡抑制蛋白(XIAP)的表达,比色法测定caspase 3活性。结果显示,与对照组相比,病毒组细胞出现凋亡形态学特征(如染色质凝集)改变,凋亡细胞数明显增多,Western blot检测结果显示XIAP表达下调,caspase 3酶活性显著增高。这些结果提示,在脐带血单核细胞向树突状细胞分化过程中,EB病毒通过上调caspase 3活性,下调XIAP表达而诱导细胞凋亡。这可能是EBV逃逸宿主免疫监视的机制之一。
To investigate the effect of Epstein-Barr virus on apoptosis during the differentiation of cord blood mononuclear cells into dendritic cells, umbilical cord blood of full-term newborns was collected under sterile conditions. Mononuclear cells were isolated and purified by rhIL-4 and rhGM- CSF induced differentiation to dendritic cells. The virus group was added with B95.8 culture supernatant on day 0 of culture. Hoechst 33258 staining was used to observe the morphological changes of the cells. The apoptotic rate was detected by Annexin V-FITC / PI double labeling flow cytometry. The expression of X-linked inhibitor of apoptosis protein (XIAP) was detected by Western blot and caspase 3 activity by colorimetric assay. The results showed that compared with the control group, the morphological changes of apoptotic cells (such as chromatin condensation) and the number of apoptotic cells in the virus group were significantly increased. Western blot results showed that the expression of XIAP was down-regulated and the activity of caspase 3 was significantly increased. These results suggest that during the differentiation of cord blood mononuclear cells into dendritic cells, Epstein-Barr virus induces apoptosis by up-regulating caspase 3 activity and down-regulating XIAP expression. This may be one of the mechanisms by which EBV escapes host immune surveillance.