热休克联合OK-432处理PANC1细胞裂解物致敏的树突状细胞体外抗癌实验  

In vitro anti-tumor assay of dendritic cells sensitized by heat shock and OK-432 treated PANC1 lysate

作　　者：刘文佳[1] 邹晓平[1] 曹俊[1] 张斌[1] 陈敏[1] 魏小雷[1] 

机构地区：[1]南京大学医学院附属鼓楼医院消化科,南京210008

出　　处：《中华胰腺病杂志》2009年第3期193-197,共5页Chinese Journal of Pancreatology

摘　　要：目的探讨热休克联合OK-432对人外周血单个核细胞（peripheral blood mononuclear cells，PBMC）来源的树突状细胞（dendritic cells，DC）体外培养的影响和对DC诱导的自身淋巴细胞抗肿瘤效果的影响。方法用细胞因子诱导外周血单核细胞转化成DC，用胰腺癌细胞株（PANC1）裂解产物（TL）致敏DC，置37、38、39、40℃热休克（HS）12h，加入体外促成熟剂OK-432，分为DC、TL—DC、HS—TL—DC、OK—TL—DC和OK—HS—TL—DC组，分别与自身淋巴细胞共培养。ELBA法检测DC上清IL-12和TNF-α含量，流式细胞仪检测细胞表面抗原CD11c、CD80、CD86、HLA—DR，TLR-4和DC成熟标志物CD83的表达，Western blotting和细胞免疫荧光法检测DC的热休克蛋白（HSP-70）表达，活细胞计数（CCK-8）法检测DC刺激自身淋巴细胞对胰腺癌细胞株PANC1、PaTu8988和胃癌细胞株SGC7901的杀伤率。结果DC体外诱导成功，HLA—DR、CD80、CD86和CD11c的表达率分别为95．1％、79．5％、80．3％和93．2％。40℃热休克12h致敏DC的TLR-4表达率从对照的2．3％增加到92．1％（P〈0．01），HSP-70表达也明显增加。CD83表达率从0．5％增加到78．7％（P〈0．01）。OK—HS—TL—DC组IL—12和TNF-α含量分别为（71．25±14．29）pg／ml和（196．59±7．47）pg／ml，较其他各组显著增加。对PANC1的杀伤率从（3．78±0．24）％提高到（72．73±4．35）％，对PaTu8988杀伤率从（4．77±0．67）％提高到（15．44±0．98）％，对SGC7901几乎没有杀伤作用。结论经热休克联合OK432处理后PANC1细胞裂解物致敏的DC生物活性增强，其刺激的自身淋巴细胞可产生高效特异的抗肿瘤免疫效应。Objective To investigate the effect of heat shock（HS） and OK-432 on in vitro culture of peripheral blood mononuclear ceils （ PBMC ） derived dendritic cells （DC） and the anti-tumor effect of host lymphocytes induced by DC. Methods PBMC was induced by cytokines to be transformed to DC, then DCs were sensitized by PANC1 pyrolysate （TL） , PANC1 lysate-pulsed DCs were exposed to HS with temperatures of 37, 38, 39, 40℃ for 12 h, then DCs were activated by Streptococcal preparation OK-432. Differently treated DCs were divided into 5 groups, which were DC, TL-DC, HS-TL-DC, OK-TL-DC and OK-HS-TL-DC group. Host lymphocytes were stimulated by different groups of DCs and co-cultured. The expressions of cell surface antigen CD11c, CD80, CD86, HLA-DR, TLR-4 and DC mature marker （ CD83 ） were detected by flow cytometry; the expression of HSP-70 in the cellular surface of DCs was examined by Western blotting and immunofluorescence. The cytotoxicity of CTL elicited by modified DCs to PANC1, PaTu8988 and SGC7901 tumor cells were measured by CCK-8 test. Results Mature DCs were obtained successfully, the expression rates of cellular surface molecules HLA-DR, CD80, CD86 and CD11c were separately 95.1% , 79.5% , 80.3% and 92.1%. The TLR-4 expression rate of DCs after 40℃ HS sensitization increased from 2.3% to 92.1% （P 〈 0.01 ）, and the expression rate of HSP-70 significantly increased also. The expression rates of OK-HS-TL- DC group over-secreted cytokines of IL-12 and TNF-α were 71.25 ± 14.29 pg/ml and （ 196.59 ±7.47 ） pg/ ml, respectively; the expression rate of CD83 increased from 0.5% to 78.7% （P 〈0.01 ） ; the killing rate on PANC1 increased from 3.78 ±0.24 to 72.73 ±4.35%, the killing rate on Patu8988 increased from （4.77 ±0.67） % to （ 15.44 ±0.98） % ; but it had no effect on SGC-7901 tumor cells. Conclusions PANC1 lysatepulsed DCs treated with heat shock and OK-432 demonstrated enhanced biological activities, and could induce host lymphocytes to highly effective and specif

关 键 词：胰腺肿瘤 树突细胞 热休克反应 

分 类 号：R73-3[医药卫生—肿瘤]