低剂量循环应用环磷酰胺联合树突状细胞疫苗对小鼠黑色素瘤的治疗效果  

Curative effect of cyclical administration of low dose cyclophosphamide combined with dendritic cells on melanoma in mice

作　　者：李卫泊[1] 尹昱[2] 张峻岭[3] 谢绍建[1] 周保军[1] 宋伟庆[1] 蔡建辉[2] 

机构地区：[1]河北医科大学第二医院胃肠外科,石家庄050000 [2]河北省人民医院康复科,石家庄050051 [3]北京大学第一医院普外科,北京100034

出　　处：《中国生物制品学杂志》2012年第6期709-713,共5页Chinese Journal of Biologicals

基　　金：河北省科技支撑计划项目(09276418D-26;10246139D);河北省医学适用技术跟踪项目(GL200938)

摘　　要：目的观察低剂量循环应用环磷酰胺(Cyclophosphamide,CTX)联合树突状细胞(Dendritic cells,DCs)疫苗对小鼠黑色素瘤的治疗效果,并探讨其机制。方法体外培养小鼠骨髓来源的DCs,制备DCs疫苗。通过皮下接种黑色素瘤B16细胞复制黑色素瘤荷瘤鼠模型,将荷瘤小鼠随机分为5组:循环CTX+DCs疫苗组、单次CTX+DCs疫苗组、单纯DCs疫苗组、单纯循环CTX组和单纯荷瘤组,治疗方式为腹腔注射CTX(100 mg/kg)后4 d,经瘤周皮下注射TL-DCs疫苗。每隔7 d治疗1次,共3次。观察肿瘤生长情况、小鼠存活状况、自身免疫病及化疗毒副反应的发生情况;于末次治疗后第5天处死小鼠,采用ELISA法检测各组小鼠血清中IFNγ水平,免疫组化法检测小鼠淋巴结内CD8阳性细胞的数量。结果循环CTX+DCs疫苗组抑瘤效果最明显(P<0.01),该组小鼠平均存活期明显高于其他各组(P<0.01),血清中IFNγ水平明显高于其他各组(P<0.01),淋巴结内CD8阳性细胞数量和平均光密度值均高于其他各组(P<0.01);应用CTX的各治疗组中均未见免疫性白斑等自身免疫病及明显化疗毒副反应。结论循环应用低剂量CTX可规律调控调节性T细胞Tregs,能显著提高DCs疫苗的疗效。Objective To observe the curative effect of cyclical administration of low dose cyclophosphamide（CTX） combined with dendritic cells（DCs） on melanoma in mice and investigate the relevant mechanism.Methods Mouse marrow-derived DCs were cultured in vitro and prepared into a vaccine（TL-DCs）.Mouse model of melanoma was copied by subcutaneous inoculation with B16 cells.The model mice were randomly divided into five groups.The mice in cyclical CTX ＋ DCs group were injected i.p.with 0.2 ml CTX（100 mg / kg） on days 7,14 and 21,then inoculated s.c.with TL-DCs at a dosage of 2 × 106 cells around the tumor on days 11,18 and 25 after tumor-bearing,while those in single CTX ＋ DCs group with CTX on day 7 and TL-DCs on days 11,18 and 25,those in DCs group with TL-DCs on days 11,18 and 25,and those in cyclical CTX group with CTX on days 7,14 and 21.However,the mice in control group were injected i.p.or s.c.with 0.2 ml physiological saline at the corresponding time points.The mice were observed for tumor growth,survival,autoimmune disease and adverse reactioin caused by chemotherapy,then killed on day 5 after the last administration and determined for serum IFNγ level by ELISA,and for CD8＋ cell count in lymph nodes by immunohistochemical assay.Results The tumor-inhibiting effect in CTX ＋ DCs group was superior to those in other groups（P 〈 0.01）,expressed as relatively long mean survival period（P 〈 0.01）,high serum IFNγ level（P 〈 0.01） as well as high CD8＋ cell count and high mean optical density（both P 〈 0.01） in lymph nodes.No autoimmune disease such as immune white spots or obvious adverse reaction to chemotherapy was observed in mice treated with CTX.Conclusions Cyclical administration of low dose CTX regulated Tregs and enhanced the curative effect of TL-DCs.

关 键 词：环磷酰胺 树突状细胞 黑色素瘤 调节性T细胞 

分 类 号：R739.5[医药卫生—肿瘤] R392-33[医药卫生—临床医学]