肝癌细胞HepG2与树突状细胞融合瘤苗体外免疫效应的实验观察  

Experimental study on the immune response of fusion tumor vaccine of HepG2 and dendritic cells in vitro

作　　者：庞业滨 崔碧玉 何坚[4] 黄馨萍 梁伟[4] 黎乐群[3] 罗小玲[2] 

机构地区：[1]广西医科大学附属肿瘤医院妇瘤科,南宁530021 [2]广西医科大学附属肿瘤医院实验研究部,南宁530021 [3]广西医科大学附属肿瘤医院肝胆外科,南宁530021 [4]广西医科大学生物靶向治疗研究中心

出　　处：《中华医学杂志》2017年第7期535-539,共5页National Medical Journal of China

基　　金：国家自然科学基金（81360347）;广西自然科学基金重点项目（2015GXNSFDA139024）;广西高校科学技术研究重点项目（ZD2014027）

摘　　要：目的制备HepG2细胞与树突状细胞（DC）融合瘤苗，检测其体外诱导的特异性抗肝癌细胞HepG2的免疫效应。方法以密度梯度离心法分离健康志愿者外周血中的单个核细胞（PBMC），以重组人粒细胞/巨噬细胞集落刺激因子（rhGM-CSF）、重组人白细胞介素4（rhIL-4）体外诱导培养DC，同时利用重组人白细胞介素2（rhIL-2）培养获得同源T淋巴细胞；采用聚乙二醇（PEG）诱导DC和HepG2融合，免疫荧光染色观察融合细胞状态，并通过流式细胞术检测融合率；使用ELISA法检测融合细胞分泌白细胞介素12（IL-12）的能力，荧光衰减法检测融合细胞刺激T淋巴细胞增殖的能力；采用ELISPOT法检测融合细胞诱导的细胞毒T淋巴细胞（CTL）释放干扰素（IFN-γ）的能力；采用乳酸脱氢酶（LDH）释放法检测融合细胞刺激产生的CTL体外特异性杀伤HepG2肝癌细胞的效果。以单纯DC、HepG2和DC混合细胞（mix DC, mDC）作为对照。结果HepG2/DC融合瘤苗体外融合率为54.5%，融合瘤苗表面高表达DC成熟分子，分别为HLA-ABC 96.7%，HLA-DR 94.5%, CD80 93.2%, CD83 90.4%，CD86 95.1%，与对照组相比差异有统计学意义（P〈0.01）；融合瘤苗刺激T淋巴细胞增殖能力显著强于对照组（P〈0.01），其分泌IL-12的水平也高于对照组（P〈0.05）；融合瘤苗诱导的CTL分泌IFN-γ的能力显著增强，特异性CTL在效靶比为30∶1和100∶1时对HepG2的杀伤率为（31.4±2.4）%和（57.6±7.3）%，比对照组杀伤能力强（P〈0.01）。结论HepG2细胞与DC能够成功融合，融合细胞能够有效提呈HepG2抗原，诱导T淋巴细胞在体外产生针对HepG2细胞的特异性免疫反应。Objective To estimate the immune response of HepG2/dendritic cell （DC） fusion cells vaccines against HepG2 cells in vitro. Methods Peripheral blood mononuclear cells （PBMCs） were isolated from healthy donors by Ficoll-Hypaque density-gradient centrifugation. Then DC were obtain from PBMCs by culturing in medium containing granulocyte macrophage colony- stimulating factor （GM-CSF） and interleukin-4 （IL-4） for 5 days. DC and HepG2 fusion cells were induced by polythyleneglycol （PEG）. The fusion cells were examined under fluorescence microscope by labeling DCs and HepG2 with green and red fluorescein, respectively, and then the fusion rates were analyzed by flow cytometry. The capacity of fusion cells to secrete interleukin （IL） -12 and stimulate the proliferation of T lymphocyte was assessed by ELISA and Flow cytometry, respectively. ELISPOT was used to assess the interferon gamma （IFN-γ） produced by cytotoxicity T lymphocyte （ CTL）, and the specific killing ability of fusion cells induee-CTL targeting HepG2 was estimated. Results The fusion rate of HepG2/DC was 54. 5%, and the fusion cells expressed a higher levels of DC mature marker CD80 and costimulatory molecules CD83, CD86 and MHC-Ⅰ , MHC-Ⅱ molecules HLA-ABC and HLA-DR than those in immature DCs （P 〈 0. 01 ）. HepG2/DC showed a greater capacity to secrete high level of IL-12 （ P 〈 0.05 ） and activate proliferation of lymphocytes in vitro, as compared with DCs alone and DCs mix HepG2 （P 〈 0. 01 ）. The HepG2/DC -activated CTL generated higher IFN-γ level and had a specific killing ability against HepG2 cells at the effecter/target ratio 30：1 （31.4% ± 2.4% ） and 100：1 （57.6% ±7.3% ） （P 〈0. 01 ）. Conclusions HepG2/DC fusion cells could efficiently stimulate T lymphocytes to generate specific CTL targeting HepG2 cells. It might be a promising strategy of immunotherapy for HCC.

关 键 词：肝癌细胞 树突状细胞 细胞融合 免疫治疗 肝细胞癌 

分 类 号：R735.7[医药卫生—肿瘤]