CAJ | 학술논문

目的：观察负载抗原的树突状细胞（DC）与细胞因子诱导的杀伤细胞（CIK ）对高表达 P‐糖蛋白（P‐gp）的多药耐药（MDR）的肝癌细胞株 HepG2／ADM 细胞的杀伤作用和机制研究。方法用常规方法诱导健康志愿者外周血中单个核细胞产生 DC 和 CIK 细胞，制备 HepG2／ADM 细胞冻融抗原后冲击 DC ，并与 CIK 细胞分别共培养24、48、72、96 h ，并将未负载抗原的 DC 和 CIK 细胞共培养作为对照。采用流式细胞术鉴定 DC 、CIK 细胞的表型，采用 CCK‐8试剂检测 HepG2／ADM 细胞的增殖活力。用 RT‐PCR 检测各组细胞内 mdr‐1 mRNA 的水平变化， Western blot 检测细胞内 P‐gp 蛋白水平的变化。结果与未负载抗原的 DC‐CIK 相比，负载抗原的 DC‐CIK 表面分子表达明显增高（P＜0．05），对 HepG2／ADM 细胞增殖活力抑制作用更加明显（P＜0．05）。 RT‐PCR 和 Western blot 结果分别显示，随着作用时间的延长，未负载抗原的 DC‐CIK 和负载抗原 DC‐CIK 的 HepG2／ADM 细胞内的mdr‐1 mRNA 和 P‐gp 蛋白水平分别都有明显的降低（P＜0．05），而且后者的抑制作用更明显（P＜0．05）。结论经抗原冲击的 DC 和 CIK 共培养可以明显提高对多药耐药 HepG2／ADM 细胞株的杀伤活性，其机制可能与抑制与MDR 密切相关的 mdr‐1基因和及其编码的 P‐gp 蛋白水平有关。
목적：관찰부재항원적수돌상세포（DC）여세포인자유도적살상세포（CIK ）대고표체 P‐당단백（P‐gp）적다약내약（MDR）적간암세포주 HepG2／ADM 세포적살상작용화궤제연구。방법용상규방법유도건강지원자외주혈중단개핵세포산생 DC 화 CIK 세포，제비 HepG2／ADM 세포동융항원후충격 DC ，병여 CIK 세포분별공배양24、48、72、96 h ，병장미부재항원적 DC 화 CIK 세포공배양작위대조。채용류식세포술감정 DC 、CIK 세포적표형，채용 CCK‐8시제검측 HepG2／ADM 세포적증식활력。용 RT‐PCR 검측각조세포내 mdr‐1 mRNA 적수평변화， Western blot 검측세포내 P‐gp 단백수평적변화。결과여미부재항원적 DC‐CIK 상비，부재항원적 DC‐CIK 표면분자표체명현증고（P＜0．05），대 HepG2／ADM 세포증식활력억제작용경가명현（P＜0．05）。 RT‐PCR 화 Western blot 결과분별현시，수착작용시간적연장，미부재항원적 DC‐CIK 화부재항원 DC‐CIK 적 HepG2／ADM 세포내적mdr‐1 mRNA 화 P‐gp 단백수평분별도유명현적강저（P＜0．05），이차후자적억제작용경명현（P＜0．05）。결론경항원충격적 DC 화 CIK 공배양가이명현제고대다약내약 HepG2／ADM 세포주적살상활성，기궤제가능여억제여MDR 밀절상관적 mdr‐1기인화급기편마적 P‐gp 단백수평유관。
Objective To observe the killing effects of the antigen‐loaded dendritic cells(DC) and cytokine‐in‐duced killer cells(CIK ) on multidrug resistance liver cancer line HepG2/ADM with highly expressed permeability glycoprotein(P‐gp) and to explore its potential mechanisms .Methods DC cells and CIK cells were induced from the peripheral blood in healthy volunteers ′by the conventional methods ,and DC cells were impacted with HepG2/ADM cells after frozen‐thawed antigen ,which were co‐cultured with CIK cells for 24 ,48 ,72 ,96 h respectively .The DC cells without antigen‐loading and CIK cells were co‐cultured as control .The cell phenotype of DC and CIK was identified by the flow cytometry ,the cell proliferation vitality of HepG2/ADM was detected by CCK‐8 ;The expression of mdr‐1 mRNA was determined by realtime‐PCR ,the P‐gp protein level was detected by Western blot .Results Compared with non‐antigen‐loaded DC‐CIK group ,the expression of surface molecules in the antigen‐loaded DC‐CIK group was significantly increased(P< 0 .05) ,the vitality of HepG2 cells was inhibited more significantly (P < 0 .05) .The RT‐PCR and Western blot results showed that with the time extension ,the expression of mdr‐1 mRNA and P‐gp protein both were significantly decreased in the above two groups respectively(P< 0 .05) ;Furthermore ,the inhibitory effects was more significant in the antigen‐loaded group(P< 0 .05) .Conclusion The co‐culture of DC and CIK after antigen impact could improve its killing activity to multidrug‐resistant HepG2/ADM cell line ,its potential mechanism may be via inhibiting the MDR closely related mdr‐1 gene expression and its encoded P‐gp protein level .