胰腺癌相关抗原MUC1与survivin mRNA联合转染树突细胞激发特异性细胞毒T淋巴细胞能力的体外研究
이선암상관항원MUC1여survivin mRNA연합전염수돌세포격발특이성세포독T림파세포능력적체외연구
In vitro study of induction of specific cytotoxic T lymphocytes by the dendritic cells co-transfected with pancreatic cancer-associated antigen MUC1 and survivin mRNA
  • 万方数据
    中华胰腺病杂志 중화이선병잡지
    CHINESE JOURNAL OF PANCREATOLOGY
    2014年 4期 217-222 ,共6页

    陈江%郭晓钟%李宏宇%邵晓东%王迪%赵佳钧%许文达

    진강%곽효종%리굉우%소효동%왕적%조가균%허문체

    树突细胞%RNA转染%胰腺肿瘤%T淋巴细胞,细胞毒性 樹突細胞%RNA轉染%胰腺腫瘤%T淋巴細胞,細胞毒性
    수돌세포%RNA전염%이선종류%T림파세포,세포독성
    Dendritic cells%RNA transfection%Pancreatic neoplasms%T-lymphocytes,cytotoxic
    目的 研究人胰腺癌MUC1与survivin mRNA联合转染树突细胞(DC)体外激发特异性细胞毒T淋巴细胞(CTL)的能力,为构建负载多抗原表位DC疫苗治疗胰腺癌提供实验依据.方法 自6例胰腺癌患者外周血单核细胞中分离、培养并鉴定DC.常规培养人胰腺癌细胞株MiaPaCa-2,采用RT-PCR方法扩增MUC1和survivin mRNA.应用电穿孔法将两种mRNA单独或联合转染DC,分别命名为DC-MUC1、DC-survivin、DC-MUC1+ survivin.采用实时定量PCR法检测DC的MUC1、survivin mRNA表达;四甲基偶氮唑蓝(MTT)法检测DC存活率;使用混合细胞培养法检测转染DC体外刺激自体T淋巴细胞的增殖能力;应用ELISA法检测转染DC体外激发抗原特异性CTL释放Th1型细胞因子IL-2、IL-10、granzyme B、IFN-γ的水平.结果 成功获得成熟的DC,成熟DC的表面标志物CD40、HLA-DR、CD83和CD86的阳性表达率分别为34.31％、50.21％、89.17％和73.62％.DC-MUC1的MUC1 mRNA表达量为36.24±5.17;DC-survivin的survivin mRNA表达量为34.53±4.02;DC-MUC1+survivin的MUC1、survivin mRNA表达量分别为31.79±4.26和14.67±2.96,显著低于单转染的DC(P值均＜0.05).DC-MUC1+ survivin的存活率呈现时间依赖性下降,96 h时的存活率显著低于单转染DC(50.21％比80％左右,P值均＜0.05).当作为刺激细胞的DC和作为效应细胞的T淋巴细胞比例为1∶10、1∶20时,DC-MUC1+ survivin刺激自体T细胞的增殖指数显著高于单转染DC,差异有统计学意义(P值均＜0.05);而比例为1∶40、1∶80时的增殖指数差异无统计学意义.当DC∶T为1∶10孵育14 d时,DC-MUC1、DC-survivin、DC-MUC1+survivin的IL-2水平分别为(892.73±32.90)、(713.62±56.37)、(1884.37±95.21) pg/ml;granzyme B水平分别为(501.62±12.30)、(203.84±12.55)、(1193.15±86.04) pg/ml;IFN-γ水平分别为(981.50±47.82)、(696.05±41.66)、(2237.94±189.55) pg/ml.DC-MUC1+ survivin显著高于单转染的DC,差异有统计学意义(P值均＜0.05);而分泌的IL-10的差异无统计学意义.结论 MUC1与survivin mRNA联合转染的DC较单一抗原转染的DC具有更强的体外特异性CTL激发能力.
    목적 연구인이선암MUC1여survivin mRNA연합전염수돌세포(DC)체외격발특이성세포독T림파세포(CTL)적능력,위구건부재다항원표위DC역묘치료이선암제공실험의거.방법 자6례이선암환자외주혈단핵세포중분리、배양병감정DC.상규배양인이선암세포주MiaPaCa-2,채용RT-PCR방법확증MUC1화survivin mRNA.응용전천공법장량충mRNA단독혹연합전염DC,분별명명위DC-MUC1、DC-survivin、DC-MUC1+ survivin.채용실시정량PCR법검측DC적MUC1、survivin mRNA표체;사갑기우담서람(MTT)법검측DC존활솔;사용혼합세포배양법검측전염DC체외자격자체T림파세포적증식능력;응용ELISA법검측전염DC체외격발항원특이성CTL석방Th1형세포인자IL-2、IL-10、granzyme B、IFN-γ적수평.결과 성공획득성숙적DC,성숙DC적표면표지물CD40、HLA-DR、CD83화CD86적양성표체솔분별위34.31％、50.21％、89.17％화73.62％.DC-MUC1적MUC1 mRNA표체량위36.24±5.17;DC-survivin적survivin mRNA표체량위34.53±4.02;DC-MUC1+survivin적MUC1、survivin mRNA표체량분별위31.79±4.26화14.67±2.96,현저저우단전염적DC(P치균＜0.05).DC-MUC1+ survivin적존활솔정현시간의뢰성하강,96 h시적존활솔현저저우단전염DC(50.21％비80％좌우,P치균＜0.05).당작위자격세포적DC화작위효응세포적T림파세포비례위1∶10、1∶20시,DC-MUC1+ survivin자격자체T세포적증식지수현저고우단전염DC,차이유통계학의의(P치균＜0.05);이비례위1∶40、1∶80시적증식지수차이무통계학의의.당DC∶T위1∶10부육14 d시,DC-MUC1、DC-survivin、DC-MUC1+survivin적IL-2수평분별위(892.73±32.90)、(713.62±56.37)、(1884.37±95.21) pg/ml;granzyme B수평분별위(501.62±12.30)、(203.84±12.55)、(1193.15±86.04) pg/ml;IFN-γ수평분별위(981.50±47.82)、(696.05±41.66)、(2237.94±189.55) pg/ml.DC-MUC1+ survivin현저고우단전염적DC,차이유통계학의의(P치균＜0.05);이분비적IL-10적차이무통계학의의.결론 MUC1여survivin mRNA연합전염적DC교단일항원전염적DC구유경강적체외특이성CTL격발능력.
    Objective To investigate the ability of induction of specific cytotoxic T lymphocytes (CTL) stimulated by dendritic cells (DCs) co-transfected with MUC1 and survivin mRNA of human pancreatic cancer,and to provide the experimental basis for the treatment of human pancreatic cancer with multi-epitope DC vaccine.Methods DCs were isolated and cultured from peripheral blood mononuclear cells (PBMCs) of 6 patients with pancreatic cancer.Human pancreatic cancer cell line MiaPaCa-2 was routinely cultured,after being transcripted and amplified by RT-PCR,MUC1 and survivin mRNA were co-transfected or individually transfected into DCs by electroporation,and they were named as DC-MUC1,DC-survivin,DC-MUC1 + survivin.The expression of MUC1 and survivin mRNA in DCs were detected by real-time PCR.The survival rate of transfected DCs were determined by MTT method.The lymphocyte proliferation ability was evaluated by mixed cell culture method.The Th1 cytokine releasing of antigen-specific CTLs were measured by ELISA assay.Results Mature DCs were obtained,the positive expression rates of surface markers CD40,HLA-DR,CD83 and CD86 were 34.31％,50.21％,89.17％ and 73.62％,respectively.The expression amount of MUC1 mRNA of DC-MUC1 was 36.24 ± 5.17,and the expression amount of survivin mRNA of DC-survivin was 34.53 ± 4.02,while the expression amounts of MUC1,survivin mRNA of DC-MUC1 + surviving were 31.79 ±4.26 and 14.67 ± 2.96,which were significantly lower than that in individual transfection group (P ＜ 0.05).The survival rate of DC-MUC1 + surviving was decreased in a time dependent manner,which was significantly lower than that in individual transfection group (about 50.21％ vs 80％ at 24 h,P ＜0.05).When DC/T cells ratio was 1∶ 10,1∶ 20,the autologous T cell proliferation index of MUC1 and survivin mRNA in co-transfection DC group was significantly higher than that in individual transfection group (P ＜ 0.05) ;when DC/T cells ratio was 1∶ 40,1∶ 80,the difference of proliferation index was not statistically significant.When DC/T cells ratio was 1∶ 10,after 14 d culture,the expressions of IL-2 in DC-MUC1,DC-survivin,DC-MUC1 + surviving were (892.73 ± 32.9),(713.62 ± 56.37),(1884.37 ± 95.21) pg/ml,and the expressions of granzyme B were (501.62 ± 12.30),(203.84 ± 12.55),(1193.15 ± 86.04) pg/ml ; and the expressions of IFN-γ were (981.50 ± 47.82),(696.05 ± 41.66),(2237.94 ± 189.55) pg/mL.The corresponding values in DC-MUC1 + surviving group were significantly higher than those in individual transfection group (P ＜ 0.05) ; while the difference of IL-10 was not statistically significant.Conclusions DCs co-transfected with MUC1 and survivin mRNA have a stronger ability to stimulate specific CTL in vitro than individual antigen loaded DCs.
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