摘要目的 探讨乳腺癌特异基因1(Bcsg1)修饰树突状细胞(DC)疫苗诱导细胞毒T淋巴细胞(CTL)对乳腺癌细胞的免疫杀伤效应及安全性.方法 将表达Bcsg1基因重组腺相关病毒(rAAV)转染外周血单个核细胞来源DC,诱导针对Bcsg1(+)乳腺癌细胞SKBR-3的靶向性CTL.流式细胞术检测成熟DC细胞表面分子及CTL活化标志;免疫荧光法检测活化后CTL分泌γ-干扰素(IFN-γ)、白细胞介素(IL)-4水平;细胞毒实验检测不同来源CTL对SKBR-3体外杀伤效应;对CTL细胞及培养液中残留DC及残留rAAV基因进行检测.结果 rAAV/Bcsg1组DC细胞高表达CD80(90.34±1.57)、CD83(85.14±1.54)、CD86(82.36±1.38)、人类白细胞抗原DR基因(HLA-DR)(78.16±1.13),与n-rAAV/Bcsg1组比较差异有统计学意义(P=0.000),rAAV/Bcsg1组DC诱导CTL活化程度明显上升;转染组CTL分泌IFN-γ[(2 616.53±163.81) pg/ml]、IL-4[(437.36±46.33) pg/ml]水平较对照组显著升高(P=0.000);在效靶比为40∶1时,对靶细胞杀伤率为(85.40±8.48)％,对非靶细胞杀伤率仅为(24.50±4.15)％;CTL制剂残留DC极少且CTL细胞及培养液未检出残留rAAV基因.结论 rAAV/Bcsg1转染DC能诱导出针对Bcsg1抗原的高效靶向杀伤性CTL,且无rAAV基因残留.

abstractsObjective To observe the immune effect of dendric cell transduced with breast cancer specific gene 1 (Bcsgl) and to conduct safety evaluation.Methods Dendritic cells originated from the peripheral blood mononuclear cells were transfected with recombinant adeno-associated virus (rAAV)containing Bcsg1 full length cDNA to generate Bcsg1 gene modified DC vaccine T lymphocytes.Those genetically modified DC vaccine repeatedly generate Bcsg1 specific cytotoxicity T lymphocytes (CTL).CTL-mediated cell lysis to mammary cancer cells line Bcsg1 (+)-SKBR-3.The surface molecular of mature DC cells and CTL activation markers were detected by Flow cytometry.The secretion levels of interferon (IFN)-γ and interleukin (IL)-4 in CTL activated by rAAV/Bcsg1-DC,Lysate-DC and n-rAAV/Bcsg1-DC were detected by flow fluorescence.rAAV gene residues were detected in mature CTL cells and culture fluid of those cells.Results After transfection,the expression of CD80 (90.34 ± 1.57),CD83 (85.14 ± 1.54),CD86 (82.36 ± 1.38),human leukocyte antigen-DR (HLA-DR)(78.16 ± 1.13) molecular markers of DC were significantly different from the control group (P =0.000)and induced CTL activation was significantly increased.In the course of CTL induction,IFN-γ [(2 616.53 ± 163.81) pg/ml] and IL-4 [(437.36 ±46.33) pg/ml] levels were significantly different from the control group (P =0.000).And rAAV/Bcsg1-DC-CTL has a stronger cytotoxic activity and higher targeting property.When the target ratio was 40∶ 1,the killing rate of target cells was (85.40 ± 8.48) ％ and on non-target cell killing rate is only (24.50 ±4.15) ％.rAAV gene residues were not detected in mature CTL cells and culture fluid.Conclusion rAAV/Bcsg1 transfected DC activate mammary cancer specific CTL.It is a safe and effective method for the treatment of tumor immune gene therapy.