CAJ | 학술논문

目的:建立丝裂霉素诱导癌细胞凋亡致敏从人外周血诱导扩增的树突状细胞的方法.设计:以癌细胞凋亡致敏树突状细胞为观察对象的随机对照实验.单位:解放军第三军医大学大坪医院野战外科研究所,解放军军事医学科学院放射医学研究所.材料:实验于1998-04/1999-05在解放军军事医学科学院放射医学研究所完成.细胞株为QBC939胆管癌细胞株,药物为抗肿瘤药物丝裂霉素.方法:从正常人外周血分离获得单核细胞,加入50μg/L重组人粒细胞集落刺激因子,1 000 U/mL白细胞介素4,隔天一次,共4次,培养第3天,加入丝裂霉素诱导凋亡的胆管癌细胞,再继续体外培养4 d后,用树突细胞富集柱收集树突状细胞.主要观察指标:①培养的树突状细胞的鉴定.②树突状细胞和坏死胆管癌细胞以及正常培养的胆管癌细胞共培养,观察其吞噬凋亡小体负载抗原.③检测不同密度的树突状细胞(1×103/孔,5×103/孔,1×104/孔)的免疫刺激活性及负载抗原后的免疫刺激活性,以单核细胞作对照组.结果:①培养、扩增得到的树突状细胞高表达共刺激分子B7和CDla,表面具有典型不规则突起.②丝裂霉素诱导癌细胞凋亡形成的凋亡小体被树突状细胞捕捉、吞噬.③负载抗原的树突状细胞其激发同种异体T淋巴细胞增殖的能力进一步增强.结论:丝裂霉素诱导癌细胞凋亡可以致敏重组人粒细胞集落刺激因子加重组人白细胞介素4从人外周血单核细胞诱导、扩增出的树突状细胞,树突状细胞可以有效提呈凋亡胆管癌细胞的抗原,可望成为有效的肿瘤抗原致敏树突状细胞的新途径.
목적:건립사렬매소유도암세포조망치민종인외주혈유도확증적수돌상세포적방법.설계:이암세포조망치민수돌상세포위관찰대상적수궤대조실험.단위:해방군제삼군의대학대평의원야전외과연구소,해방군군사의학과학원방사의학연구소.재료:실험우1998-04/1999-05재해방군군사의학과학원방사의학연구소완성.세포주위QBC939담관암세포주,약물위항종류약물사렬매소.방법:종정상인외주혈분리획득단핵세포,가입50μg/L중조인립세포집락자격인자,1 000 U/mL백세포개소4,격천일차,공4차,배양제3천,가입사렬매소유도조망적담관암세포,재계속체외배양4 d후,용수돌세포부집주수집수돌상세포.주요관찰지표:①배양적수돌상세포적감정.②수돌상세포화배사담관암세포이급정상배양적담관암세포공배양,관찰기탄서조망소체부재항원.③검측불동밀도적수돌상세포(1×103/공,5×103/공,1×104/공)적면역자격활성급부재항원후적면역자격활성,이단핵세포작대조조.결과:①배양、확증득도적수돌상세포고표체공자격분자B7화CDla,표면구유전형불규칙돌기.②사렬매소유도암세포조망형성적조망소체피수돌상세포포착、탄서.③부재항원적수돌상세포기격발동충이체T림파세포증식적능력진일보증강.결론:사렬매소유도암세포조망가이치민중조인립세포집락자격인자가중조인백세포개소4종인외주혈단핵세포유도、확증출적수돌상세포,수돌상세포가이유효제정조망담관암세포적항원,가망성위유효적종류항원치민수돌상세포적신도경.
BACKGROUND: Dendritic cells play an important role in antigen present in vivo, and the mechanism of tumor cells in escaping the antigen presentation of dendritic cells existed in the patients with tumor.OBJECTIVE: To sensitize dendritic cells from human peripheral blood with apoptotic hepatoma cells induced by mitomycin.DESIGN: A randomized control trial by taking apoptotic hepatoma cell sensitized dendritic cells as the observed subjects.SETTINGS: Institute of Field Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA; Institute of Radiation Medicine,Chinese PLA Academy of Military Medical Sciences.MATERIALS: The experiments were carried out in the Institute of Radiation Medicine, Chinese PLA Academy of Military Medical Sciences from April 1998 to May 1999. The cell strain was the QBC939 bile duct cancer cell strain, and mitomycin was used as the antitumor drug.METHODS: Mononuclear cells were isolated from the peripheral blood of normal people, 50μg/L granulocyte-macrophage colony stimulating factor(GM-CSF) and 1 000 U/mL interleukin-4 (IL-4) were added, once every other day for 4 times. On the 3rd day of culture, the apoptotic bile duct cancer cells induced by mitomycin was added, and then cultured in vitro for 4 days, finally the dendritic cells were collected.MAIN OUTCOME MEASURES: ① The identification of the cultured dendritic cells was observed; ② The dendritic cells were co-cultured with necrotic and normally cultured bile duct cancer cells respectively, and the phagocytized apoptotic body loaded antigens were observed; ③ The immunostimulatory activity of dendritic cells (1×103, 5×103 and 1×104/well)and that after loaded by antigen were detected, and the mononuclear cells were taken as controls.RESULTS: ① The cultured and amplified dendritic cells expressed high levels of costimulatory molecules of CD1a and B7, and there were typical irregular processes on the surface. ② The tumor cells formed apoptotic bodies when they were induced by mitomycin, which were arrested and phagocytized by dendritic cells. ③ The ability of the antigen loaded dendritic cells in stimulating the proliferation of allogenic lymphocyte T was further enhanced.CONCLUSION: The apoptotic tumor cells induced by mitomycin can induce the mononuclear cells from human peripheral blood differentiating into the dendritic cells with the concomitance of GM-CSF and recombinant IL-4 and amplify dendritic cells. Meanwhile, the dendritic cells can effectively present the antigens of apoptotic bile duct cancer cells, and it probably becomes a new effective approach for tumor antigen to sensitize dendritic cells.