CAJ | 학술논문

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pCDNA3．1(+)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。
목적：：재세포수평상분석고표체OAZI-1적소서B16-F1세포능부활화항원제정세포,촉진항원제정세포대종류적탄서이급항원체정작용。방법：전염중조질립후,용RT-PCR화Western blot기술사선획득고표체OAZI-1적소서흑색소류B16-F1종류세포(B16/OAZI-1),동시구건전염공재체질립pCDNA3．1(+)적소서흑색소류B16-F1종류세포(B16/3．1)용작실험대조。제비BALB/c소서복강거서세포、비림파세포화골수래원적수돌상세포( DC),장B16/OAZI-1화B16/3．1분별여소서복강거서세포화DC안조1∶5화1∶1적세포개수비례혼합배양4 h,류식세포술검측거서세포화DC대종류세포적탄서솔。장B16/OAZI-1화B16/3．1분별여소서DC안조1∶5적세포개수비례혼합배양24 h후,류식세포술검측DC표면분자CD40、CD80화CD86표체적변화。장경종류세포활화적DC여소서비림파세포혼합배양24 h후,ELISA검측세포상청중IFN-γ적함량。결과：거서세포화DC대B16/OAZI-1세포탄서솔분별위24．7%화53．9%,여B16/3．1세포(8．2%화13．8%)상비유교현저성차이。여B16/3．1세포상비,DC세포여B16/OAZI-1세포혼합배양24 h후,성숙DC세포표면분자표지CD40、CD80、CD86적표체분별종24．2%、20．8%화16．4%증가도46．8%、32．5%화36．1%(P<0．05)；경B16/OAZI-1세포활화적DC여소서비림파세포혼합배양후,세포상청중IFN-γ적함량위32．9 pg/ml,여B16/3．1세포처리적DC상비(15．1 pg/ml)유현저성차이(P<0．05)。결론：고표체OAZI-1적종류세포능경유효지피거서세포화DC세포탄서,차저일과정능유도DC세포성숙활화,성숙적DC세포장종류항원체정급T림파세포병유도T림파세포활화,종이격활궤체항종류면역응답。
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 pcDNA3. 1(+)/OAZI-1 was transfected into B16-F1 cells by Li-pofectamine2000 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 pcDNA3. 1(+) (B16/3. 1) were used as the control cells in this experiment. B16-F1 cells and macrophages were co-cultured 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-γcontent 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. And this process can induce the maturation and activation of DCs. Matured DC could induce T cell activation and then activate the anti-tumor immune response.