热休克瘤细胞裂解物冲击的异基因树突状细胞促进抗肿瘤免疫  

Allogeneic dendritic cell vaccine pulsed with heat shocked tumor cell lysate can enhance antitumor immunity

作　　者：尤健[1] 王长利[1] 郝希山[1] 

机构地区：[1]天津医科大学附属肿瘤医院胸部肿瘤外科,300060

出　　处：《中华医学杂志》2007年第39期2785-2790,共6页National Medical Journal of China

基　　金：天津市自然科学基金(06YFJMJC08100)

摘　　要：目的探讨热休克瘤细胞裂解物冲击的异基因树突状细胞(DC)抗肿瘤免疫效应。方法应用热休克(43℃,1h)小鼠 Lewis 肺癌细胞(LLC)裂解物(LhTCL)冲击的小鼠骨髓细胞衍生的树突状细胞(DC)作为瘤苗(DC/LhTCL)。应用 Western 印迹检测可诱导 HSP70表达。用 FITC-标记的 LhTCL 和 CFSE-标记的异基因 DC 分别进行体外吞噬和体内示踪实验,藉酶联免疫吸附(ELISA)、流式细胞术(FCM)和共聚焦荧光显微镜观察分析不成熟异基因 DC 体外摄取 LhTCL 对其表型和 IL-12产生的影响以及受鼠接种局部引流淋巴结DC(LD-DC)对异基因 DC 的摄取。然后,通过免疫接种的小鼠脾细胞体外诱生 CTL 的杀瘤活性(LDH法)和产生的 Th-1相关细胞因子检测,LLC移植瘤的免疫预防和早期带瘤鼠的治疗实验以评价 C57BL小鼠接种异基因或同系 DC/LhTCL 瘤苗的产生的抗肿瘤免疫效应。结果 (1)热休克增加 LLC 胞浆内可诱导 HSP70表达。(2)小鼠不成熟异基因 DC 能充分摄取 LhTCL 并促进其 DC 成熟(增加 DC 表面共刺激分子表达和 IL-12分泌)。(3)皮下注射 LhTCL 冲击的异基因 DC 观察到有促进受鼠 DC 摄取异基因 DC 的效应。(4)LhTCL 冲击的异基因 DC 免疫的 C57BL 小鼠脾细胞能特异性杀伤活 LLC,分泌 Th-1相关的细胞因子(高水平IFN-γ和低水平 IL-4和 IL-10)。(5)C57BL 小鼠 LLC 移植瘤免疫保护实验和治疗实验均证实,免疫接种异基因 DC/LhTCL 瘤苗能够阻止或延缓 LLC 肿瘤的生长,其产生的免疫保护效应要比同系 DC/LhTCL 瘤苗强。结论接种热休克处理的肿瘤裂解物冲击的异基因树突状细胞瘤苗有促进抗肿瘤免疫的效应。Objective To explore whether the allogeneic dendritic cells-pulsed with heat shock protein （HSP）-70-rich tumor cell lysate can enhance the antitumor immunity. Methods Mouse Lewis lung cancer cells were dipped into water of the temperature of 43℃ for 1 h so as to undergo heat shock. C.57BLK^b） to elicit increased hsp70 expression, The LLCs were lysed by freeze thawing and the supernatant was collected （LhTCL）. Dendritic cells （DCs） were isolated from allogeneic inbred mice （615Kk or BALB/CKd） bone marrow （ Bm-DCs）, and were pulsed with the LhTCL that underwent heart shock. The phenotypes of the Bm-DCs were analyzed by flow cytometry （ FCM）. The level of IL-12 was detected by ELISA. The Bm-Dcs of Balb/c mice that were pulsed by LhTCL or not, were injected into the foot pads of C57BL mice as vaccine, 3 d later the lymph nodes in the drainage area of popliteal fossa were taken out to make single cell suspension to undergo immunohistochemistry. Inducible hsp70 in LhTCL was assayed by Western Blotting. The ability of taking up LhTCL by immature DC was evaluated by phagocytosis experiment in vitro and the uptake of CFSE labeled allogeneic DC by recipient DC was evaluated by tracing experiment in vivo. The supernatant cytokine release was determined by sandwich ELISA. Double stained DC was confirmed by FCM and confocal microscopy. Cytotoxic activity was assessed by LDH release assay. The immuno-effect of vaccine was valued by immunoprophylaxis in LLC transplanted model and therapy experiment of LLC-bearing mice of early stage. Result 43℃ heating significantly increased the expression ofinducible hsp70 in the LLCs. The allogeneic immature DC s showed full ability to uptake LhTCL and enhanced the expression of CD86 molecules on DC surface and IL-12 secretion, showing that they promoted the maturation of DCs. Subcutaneous injection of allogeneic DC/ LhTCL promoted the uptake of allogeneic DC by recipient DC. The spleen cells from the C57BL mice immunized with allogeneic DC/LhTCL vaccine specifi

关 键 词：树突细胞 热休克蛋白质类 肺肿瘤 抗肿瘤免疫 

分 类 号：R730.5[医药卫生—肿瘤]