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目的探讨以乳腺癌特异抗原BA46基因转导树突状细胞(DC)治疗乳腺癌的可行性。方法取健康人外周血,采用密度梯度离心的方法分离外周血单个核细胞(DC前体细胞),以AIM-V培养基于6孔板培养,贴壁5h,轻轻洗去悬浮细胞(T细胞,冻存备用),取贴壁细胞,将细胞分为基因转染组及对照组,基因转染组感染携带BA46基因的重组腺相关病毒rAAV/BA46/Neo,对照组以293细胞冻融液刺激,两组细胞均采用重组人粒细胞巨噬细胞集落刺激因子(GM-CSF)、白细胞介素4(IL-4)、肿瘤坏死因子-α(TNF-α)诱导DC前体细胞成熟。第7天,收集悬浮细胞(DC),显微镜观察细胞形态,流式细胞仪分析DC的表面标志CD80、CD86、CD40的表达情况;另取该DC与T细胞按1∶20比例混合,含5%人血清蛋白的AIM-V为培养基,同时加入IL-2与GM-CSF,共育7d,诱导获得细胞毒性T淋巴细胞(CTL),取BA46阳性的乳腺癌细胞株Hs578T为靶细胞,采用51Cr释放法测量杀伤效率,同时以流式细胞仪检测CTL群体中CD8/CD4和CD8/CD56的比值。结果BA46基因转导的DC表面标志CD80、CD86、CD40的表达均明显高于对照组DC,所激活的T细胞对BA46阳性的乳腺癌细胞株Hs578T有很好的杀伤效果,此杀伤具有抗原特异性和MHC限制性,且该T细胞中CD8/CD4和CD8/CD56的比值均明显高于对照组(细胞裂解物冲击DC所激活的T细胞)。结论BA46基因转染成功制备DC,并诱导特异的CTL,为乳腺癌的DC基因转染疗法打下基础。
Objective To investigate the feasibility of treating breast cancer by transduction of dendritic cells (DCs) with BA46 gene, a specific antigen of breast cancer. Methods Peripheral blood mononuclear cells (DCs) were isolated by density gradient centrifugation and cultured in 6-well plates with AIM-V culture for 5h. The cells were gently washed away from the T cells The cells were divided into gene transfection group and control group. The gene transfection group was infected with recombinant adeno-associated virus rAAV / BA46 / Neo carrying BA46 gene, and the control group was infected with 293 cells freeze-thaw solution (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-α (TNF-α) were used to induce DC precursor cell maturation in both groups. On the 7th day, the cells were harvested, and the morphology of the cells was observed under a microscope. The surface markers of CD80, CD86 and CD40 were analyzed by flow cytometry. The DCs and T cells were mixed 1:20, % AIM-V of human serum albumin was used as culture medium, IL-2 and GM-CSF were added together for 7 days to induce cytotoxic T lymphocytes (CTLs), and a BA46-positive breast cancer cell line Hs578T was used as a target cell. The cytotoxicity was measured by 51Cr release method. Meanwhile, the ratio of CD8 / CD4 and CD8 / CD56 in CTL population was detected by flow cytometry. Results The expression of CD80, CD86 and CD40 on DCs transduced with BA46 gene was significantly higher than that of control group. The activated T cells had a good killing effect on BA46 positive breast cancer cell line Hs578T, which had antigen specificity Sexual and MHC-restricted, and the ratio of CD8 / CD4 and CD8 / CD56 in the T cells were significantly higher than that of the control group (cell lysate impacted T cells activated by DCs). Conclusions The successful transfection of BA46 gene into DCs and the induction of specific CTLs lay the foundation for DC gene transfection in breast cancer patients.