低危型HPV-11E6在小鼠骨髓源树突细胞中的定位表达及其诱发凋亡的研究

Location of Low risk HPV-11E6 in bone marrow-derived dendritic cells and its effect on apoptosis

目的采用绿色荧光蛋白表达系统追踪低危型HPV-11E6蛋白在树突细胞(dentritic cell,DC)内的表达,探讨低危型HPV-11E6在树突细胞中的定位,观察低危型HPV-11E6蛋白诱导的树突细胞凋亡。方法构建真核表达载体pGFP-11E6,转染小鼠骨髓源树突细胞,荧光显微镜动态观察E6蛋白的定位和表达水平。采用流式细胞术检测转染后24 h低危型HPV-11E6蛋白的凋亡。结果树突细胞在分别转染pGFP-11E6和p EGFP-C1质粒后,GFP-11E6主要定位于细胞质内;而对照组的只表达GFP的蛋白则均匀分布于树突细胞。蛋白表达水平的检测表明,在转染树突细胞12 h,GFP-11E6和GFP蛋白开始表达(荧光强度分别为73.22,84.34),转染24 h蛋白表达均到达高峰(荧光强度分别为98.25,126.77),转染48 h蛋白表达荧光强度分别为87.46和103.56,转染72 h蛋白表达荧光强度分别为72.11和97.45。Annexin-Ⅴ/PI流式细胞仪检测可见,GFP-11E6转染的细胞发生了凋亡。结论低危型HPV-11E6主要定位于树突细胞质内,并且可以诱导树突细胞凋亡。

Objective To investigate the location of GFP-11E6 in bone marrow dendritic cells using green fluorescence expression system and the apoptosis of GFP-11E6-induced dendritic cells.Methods The plasmid GFP-11E6 was constructed and transfected into dendritic cells from mouse bone marrow.The location and expression level of E6 protein were observed under fluorescent microscope.Flow cytometry was used to detect the apoptosis in GFP-11E6-expressed dendritic cells.Results GFP-11E6 was predominantly expressed in the cytoplasm of dendritic cells after transfection,whereas the GFP control was located in both cytoplasm and nuclei by fluorescent microscope.The expression fluorescence intensities of GFP-11E6 and GFP were 73.22,84.34 at 12 h after transfection,and they increased to the highest level（98.25,126.77） at 24 h after transfection.Then the fluorescence intensities decreased to 87.46,103.56 at 48 h after transfection,and 72.11,97.45 at 72 h after transfection.Apoptosis was increased in HPV-11E6-expressed dendritic cells from 24 h after transfection.Conclusion The low risk HPV11-E6 is mainly located in cytoplasm in dendritic cells and can further induce apoptosis.