中文摘要：

目的：在细胞水平上分析高表达OAZI-1的小鼠B16-F1细胞能否活化抗原提呈细胞,促进抗原提呈细胞对肿瘤的吞噬以及抗原递呈作用。方法：转染重组质粒后,用RT-PCR和Western blot技术筛选获得高表达OAZI-1的小鼠黑色素瘤B16-F1肿瘤细胞（B16/OAZI-1）,同时构建转染空载体质粒p CDNA3.1（＋）的小鼠黑色素瘤B16-F1肿瘤细胞（B16/3.1）用作实验对照。制备BALB/c小鼠腹腔巨噬细胞、脾淋巴细胞和骨髓来源的树突状细胞（DC）,将B16/OAZI-1和B16/3.1分别与小鼠腹腔巨噬细胞和DC按照1∶5和1∶1的细胞个数比例混合培养4 h,流式细胞术检测巨噬细胞和DC对肿瘤细胞的吞噬率。将B16/OAZI-1和B16/3.1分别与小鼠DC按照1∶5的细胞个数比例混合培养24 h后,流式细胞术检测DC表面分子CD40、CD80和CD86表达的变化。将经肿瘤细胞活化的DC与小鼠脾淋巴细胞混合培养24 h后,ELISA检测细胞上清中IFN-γ的含量。结果：巨噬细胞和DC对B16/OAZI-1细胞吞噬率分别为24.7%和53.9%,与B16/3.1细胞（8.2%和13.8%）相比有较显著性差异。与B16/3.1细胞相比,DC细胞与B16/OAZI-1细胞混合培养24 h后,成熟DC细胞表面分子标志CD40、CD80、CD86的表达分别从24.2%、20.8%和16.4%增加到46.8%、32.5%和36.1%（P〈0.05）;经B16/OAZI-1细胞活化的DC与小鼠脾淋巴细胞混合培养后,细胞上清中IFN-γ的含量为32.9 pg/ml,与B16/3.1细胞处理的DC相比（15.1 pg/ml）有显著性差异（P〈0.05）。结论：高表达OAZI-1的肿瘤细胞能更有效地被巨噬细胞和DC细胞吞噬,且这一过程能诱导DC细胞成熟活化,成熟的DC细胞将肿瘤抗原递呈给T淋巴细胞并诱导T淋巴细胞活化,从而激活机体抗肿瘤免疫应答。

英文摘要：

Objective： To analyze,at cellular level,whether the mouse B16-F1 melanoma cells with OAZI-1 overexpression could activate antigen-presenting cells and promote the phagocytotic and antigen-presenting efficiencies of mouse peritoneal macrophage and bone marrow derived DC on tumor cells. Methods： The plasmid pc DNA3. 1（ ＋） / OAZI-1 was transfected into B16-F1 cells by Lipofectamine2000 reagent. The positive clones with OAZI-1 overexpression（ B16 / OAZI-1） were identified by Western blot assay and RT-PCR. Macrophages from abdominal cavity and DC from bone marrow were collected from BALB / c mouse. The B16-F1 cells transfected with the pc DNA3. 1（ ＋）（ B16 /3. 1） were used as the control cells in this experiment. B16-F1 cells and macrophages were cocultured for 4 h at a 1∶ 5 ratio and DC were co-cultured with B16-F1 cells at 1∶ 1 ratio for 4 h. And then the phagocytotic efficiencies were assayed by flow cytometry. DC were co-cultured with B16-F1 cells at 1∶ 1 ratio for 24 h and then the expression of mature DC surface marker molecules CD40,CD80,CD86 were determined by flow cytometry. The DC activated by the tumor cells were co-cultured with mouse spleen lymphocytes for 24 h,and then IFN-γ content in culture medium was analyzed by ELISA. Results： Phagocytotic assay showed that,compared to the control cells,the OAZI-1 overexpression in B16-F1 cells significantly enhanced the engulfment of B16-F1 cells by macrophages（ 24. 7% vs 53. 9%） and DC（ 8. 2% vs 13. 8%）. When DC were co-incubated with OAZI-1 overexpressed B16-F1 for 24 h,the expression levels of CD40,CD80,CD86 on the DC surface,which were the molecular markers for matured DC,increased from 24. 2%,20. 8% and 16. 4% to 46. 8%,32. 5% and 36. 1% respectively. Co-culture of tumor-activated DC with the spleen lymphocytes resulted in an increased IFN-γ content in the culture medium（ 32. 9 pg / ml vs 15. 1 pg / ml）. Conclusion： The tumor cells with OAZI-1 overexpression can be engulfed more efficiently by macrophages and DC.