β-榄香烯联合DC/DRibble疫苗治疗小鼠肝癌免疫机制研究  

Treatment of Hepatic Cancer in Mice by β-elemene Combined DC/Dribble Vaccine: an Immune Mechanism Research

作　　者：倪菲菲[1] 刘亚军[2] 周浩[3] 林琳[4] 刘增巍[2] 沈洪[1,2] 王立新[3] 

机构地区：[1]南京中医药大学第一临床医学院,南京210029 [2]江苏省中医院消化科,南京210029 [3]东南大学医学院病原生物学与免疫学系,南京210009 [4]江苏省中西医结合医院,南京210000

出　　处：《中国中西医结合杂志》2013年第2期214-219,共6页Chinese Journal of Integrated Traditional and Western Medicine

基　　金：国家自然科学基金资助项目(No.30771999;30972783);江苏省中医药领军人才培养资助项目(No.LJ200901);江苏省中医院院级课题资助项目(No.Y11004)

摘　　要：目的观察β-榄香烯联合DC/DRibble疫苗对小鼠肝癌的治疗作用,探讨β-榄香烯抗肿瘤的免疫机制。方法制备Balb/c小鼠脾脏来源的DC并鉴定其表型,以Balb/c小鼠来源的肝癌细胞株BNL1MEA.7R.1(简称BNL)为靶细胞,募集富含肿瘤"信息"的抗原载体—自噬小体,制备DC/DRibble疫苗。预先免疫小鼠,分设对照组、β-榄香烯组、疫苗组、联合组,对照组皮下、腹腔注射PBS,β-榄香烯组和联合组连续7天腹腔注射β-榄香烯50mg/(kg·d),疫苗组和联合组分别在第1天淋巴结注射疫苗,第3、5天皮下注射疫苗,第10天处死小鼠,无菌获得小鼠脾脏,制备悬液,分设不加刺激和Dribble刺激组,孵育72h,ELISA法检测上清中IFN-γ的含量。另外给予联合组小鼠脾细胞不同刺激,分设对照组、DRibble组、DC组、疫苗组,孵育72h,ELISA法检测上清中IFN-γ的含量。建立小鼠肝癌荷瘤模型,分为对照组、β-榄香烯组、疫苗组、联合组,治疗方式同上免疫方式,观察各组小鼠的肿瘤大小及生存期,第23天处死小鼠,剥取肿瘤组织经HE染色,光学显微镜观察肿瘤组织病理形态学表现。结果体外检测发现不同方式免疫后小鼠中,联合组IFN-γ分泌量明显高于其他组(P<0.01),β-榄香烯组、疫苗组高于对照组(P<0.01);给予免疫后小鼠脾细胞不同刺激,发现疫苗组、Dribble组均能刺激脾细胞分泌大量IFN-γ(P<0.01),当β-榄香烯刺激浓度10μg/mL时,IFN-γ分泌明显增多(P<0.01);体内观察发现联合组小鼠肿瘤生长速度明显减慢,瘤表面积、生存期与其他组相比均有统计学差异(P<0.01),肿瘤组织HE染色可见周围结缔组织包裹紧密完整,大量炎性细胞浸润。结论β-榄香烯联合DC/DRibble疫苗能够诱导特异性免疫细胞分泌细胞因子功能增强,从而发挥抗肿瘤作用,其免疫效应基础可能与增强DC抗原递呈功能有关。Objective To observe the therapeutic effects of β-elemene combined DC/Dribble vaccine in treating mice with hepatic cancer， thus exploring their anti-tumor mechanisms. Methods Dentritic cells were derived from Balb/c mice′s spleen and their phenotypes were identified. Using hepatic cancer cell line BNL1MEA.7R.1 （abbreviated as BNL） originated from Balb/c mice as target cell， DC/Dribble vaccine was prepared via raising the antigen representing carrier autophagosomes （DRips in Blebs， DRibbles）， which were rich in tumor antigen information. The mice previously immunized were divided into 4 groups， i.e.， the control group， the β-elemene group， the vaccine group， and the combined group. The PBS was subcutaneously and intraperitoneally injected to mice in the control group. The β-lemene was intraperitoneally injected at the daily dose of 50 mg/kg to mice in the β-elemene group and the combined group for 7 successive days. DC/Dribble vaccine was injected into the lymph node of mice in the vaccine group and the combined group on the 1st day， and DC/Dribble vaccine was subcutaneously injected on the 3rd day and the 5th day. All the mice were sacrificed on the 10th day. Their spleens were obtained sterilely， and the suspension was incubated with or without Dribble. The cells were inoculated for 72 h. The contents of IFN-γ in the supernatant were measured by ELISA. In addition， the spleen cells obtained from the combined group were incubated with different stimulations for 72 h， which were then divided into the control group， the DRibble group， the DC group， and the DC/Dribble vaccine group. The supernatant of cultured cells were collected and the contents of IFN-γ were measured by ELISA. The liver tumor-bearing mouse model was established， and then the BNL bearing mice were randomly divided into 4 groups， i.e.， the control group， the β-elemene group， the vaccine group， and the combined group. The treatment ways were the same as the immune ways. The tumor size

关 键 词：Β-榄香烯 肝癌 树突状细胞 自噬小体 

分 类 号：R735.7[医药卫生—肿瘤]