Runx2-树突状细胞基因疫苗诱导特异性细胞免疫杀伤三阴乳腺癌  

Vaccine of Runx2-dendritic Cells Can Induce Specific Lethal Effect of Triple Negative Breast Cancer

作　　者：汤谧[1] 张翘楚[1] 张鹏[1] 刘瑞磊[1] 姜华[1] 黄勇[1] 

机构地区：[1]中山大学附属第三医院甲状腺乳腺外科,广东广州510630

出　　处：《中山大学学报（医学科学版）》2015年第3期377-384,共8页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省自然科学基金(2014A030313193)

摘　　要：【目的】探讨Runx2基因在乳腺癌中分布及Runx2-树突状细胞(DC)疫苗能否在体外诱导产生针对三阴乳腺癌的特异性抗肿瘤效应。【方法】RT-PCR、免疫细胞化学和Westernblot检测Runx2在三阴乳腺癌、非三阴乳腺癌和正常乳腺细胞中表达;提取健康人外周血并分离DC,流式检测DC表面标志;制备Runx2过表达慢病毒,转染DC并分为转染组、阴性对照组及空白组,荧光显微镜、PCR和Westernblot检测各组转染效率;将各组转染细胞与T淋巴细胞共培养,ELISA检测IL-12、IFN-γ的分泌量,MTT检测疫苗对三阴乳腺癌的特异性杀伤作用。【结果】PCR结果示Runx2在MDA-MB-231、MCF7及MCF10A中ΔCT值分别为10.37±0.61、11.86±0.59、12.97±0.06(P<0.05),免疫细胞化学及Western显示MDA-MB-231中Runx2蛋白呈强阳性,在MCF7及MCF10A中仅为弱阳性;本研究制备Runx2慢病毒在转染DC后可以表达丰富绿色荧光,证实Runx2基因成功转导入成熟DC中。转染组与T细胞共培养后分泌IL-12、IFN-γ量(pg/m L)分别为170.33±5.03、517.15±5.88,而阴性对照组的分泌量仅为72.30±3.05、171.57±1.37(P<0.05),同时转染组诱导的细胞毒性T细胞对三阴乳腺癌杀伤率(%)为60.02±3.51,较对照组(25.57±3.64)增强(P<0.05)。【结论】Runx2可能为三阴乳腺癌治疗新靶点,并能成功转导Runx2至DC中制备Runx2-DC疫苗,增强DC对三阴乳腺癌的靶向性,从而高效诱导该疫苗在体外对三阴乳腺癌的特异性杀伤作用。[Objective] To study the character of Runx2 gene in breast cancer and whether the Runx2-dendritic cell（DC） vaccine can induce specific antitumor effect on triple negative breast cancer in vitro.[Methods] The expression of Runx2 in triple negative breast cancer,non-triple negative breast cancer and normal breast cells were analyzed by RT-PCR,Immunocytochemistry and Western blot.DC were isolated and cultured from peripheral blood of healthy donors,the surface maker CD11c,CD80,CD86 and HLA-DR were analyzed with flow cytometry.Runx2 lentivirus were prepared to infect DC and divided in transfected group,negative group and blank group.Fluorescence microscope,PCR and Western blot tested its transfection efficiency of all groups.DC and T cells were cultured together to induce CTL,ELISA was used to detect the secretion of IL-12 and IFN-γ.The killing ability of CTL were calculated by MTT.[Results] PCR results showed the ACT value of Runx2 in MDA-MB-231,MCF7 and MCF10A were 10.37 ± 0.61,11.86 ± 0.59,12.97 ± 0.06 （P ＜ 0.05） respectively.Both Immunocytochemistry and Western blot showed Runx2 were strongly positive in MDA-MB-231,but weaker in MCF7 and MCF10A.In this study,Runx2-lentivirus were prepared and transfected DC.Rich green flurescent confirmed that the Runx2 gene were successfully turned into mature DC.Then the transfected DC were co-cultured with T cells,and the secretion of IL-12、IFN-γ （pg/mL） were measured as 170.33 ± 5.03,517.15 ± 5.88.While the secretion of IL-12 and IFN-γwere 72.30 ± 3.05 and 171.57 ± 1.37 （P ＜ 0.05） in non-transfected group.And the killing ratio of cytotoxic T cells of transfected group on triple negative breast cancer was 60.02 ±-3.51 compared with 25.57 ± 3.64 （P ＜ 0.05） of the control group.[Conclusions] Runx2 maybe a new target for triple negative breast cancer treatment.Runx2 can transduce DC successfully and the vaccine induce specific CTL and strong cytotoxicity against triple negative breast cancer.

关 键 词：RUNX2 树突状细胞 三阴乳腺癌 基因疫苗 

分 类 号：R73[医药卫生—肿瘤]