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目的探讨以腺相关病毒为载体,乳腺癌抗原BA46基因转染树突状细胞(DC)诱导特异细胞免疫的可行性。方法取健康人外周血,采用密度梯度离心法分离外周血单个核细胞(DC前体细胞),将细胞分为基因转染组及对照组,基因转染组以重组腺相关病毒rAAV/BA46/Neo转染,对照组以293细胞裂解物冲击,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DC,取该DC与T细胞按比例混合,诱导细胞毒性T淋巴细胞(CTL),用3H-TdR掺入法检测DC刺激自体淋巴细胞增殖能力;以流式细胞仪分析CTL细胞中IFN-γ、IL-4、CD4、CD8、CD25、CD69的表达情况,同时取BA46阳性的乳腺癌细胞株Hs578T为靶细胞,采用51Cr释放法检测杀伤效率。结果BA46基因转染组DC具备较强的刺激淋巴细胞增殖的能力,所诱导的CTL高表达CD8、CD69和IFN-γ,对BA46阳性的靶细胞有较强的杀伤作用,该杀伤具有BA46抗原特异性和MHC限制性。结论乳腺癌BA46基因转染DC成功诱导特异的细胞免疫,为基因修饰细胞治疗乳腺癌打下实验室基础。
Objective To investigate the feasibility of using adeno-associated virus as a carrier and dendritic cells (DCs) transfected with the BA46 gene of breast cancer to induce specific cellular immunity. Methods Peripheral blood mononuclear cells (DCs) were isolated by density gradient centrifugation. The cells were divided into gene transfection group and control group. The gene transfection group was transfected with recombinant adeno - associated virus rAAV / BA46 / Neo were transfected and the control group was challenged with 293 cell lysate. Both groups were treated with GM-CSF, IL-4 and TNF- TNF-α) induces DC precursor cell maturation. On day 7, DCs were harvested and mixed with DCs and T cells in proportion to induce cytotoxic T lymphocytes (CTLs). The ability of DCs to stimulate autologous lymphocyte proliferation was examined by 3H-TdR incorporation assay. CTLs were analyzed by flow cytometry The expression of IFN-γ, IL-4, CD4, CD8, CD25 and CD69 in the cells was detected. At the same time, H4678T cells were taken as target cells and the killing rate was measured by 51Cr release assay. Results DCs with BA46 gene had a strong ability of stimulating lymphocyte proliferation. CTLs induced high expression of CD8, CD69 and IFN-γ, and had a stronger killing effect on BA46-positive target cells. The cytotoxicity of BA46 gene with BA46 antigen Specificity and MHC restriction. Conclusions The successful transfection of BA46 gene into breast cancer induces specific cellular immunity and lays a laboratory foundation for genetically modified cells to treat breast cancer.