摘要
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 study the feasibility of transfecting breast cancer BA46 gene into dendritic ceils （DCs） using adeno-associated virus （AAV） to induce specific cellular immunity. Methods Mononuclear cells （DC precursor） were isolated from the peripheral blood of healthy donors by density gradient centrifugation and infected with rAAV/BA46/Neo virus stock （transfection group） or pulsed with 293 cell lysate （control group）. In both groups, maturation of the DC precursor was induced by granulocyte-macrophage colony-stimulating factor （GM-CSF）, interleukin-4 （IL-4） and tumor necrosis factor-α（TNF-α）. On day 7, the DCs were collected and mixed with T cells at the ratio of 1 to 20 to induce cytotoxic T lymphocytes （CTL）. The capacity of the DCs in stimulating T lymphocyte proliferation was assessed using 3H-thymidine incorporation assay. The expressions of interferon-γ（IFN-7）, IL-4, CD4, CDS, CD25 and CD69 in the CTLs were analyzed with cytometry, and the cytotoxicity of the CTLs was evaluated with ^51Cr-release assay using BA46-positive breast cancer cell line Hs578T as the target. Results The DCs transfected with BA46 gene exhibited potent capacity to stimulate T lymphocyte proliferation. The CTL population induced by the transfected DCs expressed high levels of CD8, CD69 and IFN-γ and showed strong cytotoxicity against BA46-positive breast cancer cell line Hs578T, which was BA46 antigen-specific and MHC-limited. Conclusion The success in BA46 gene transfer in the DCs that induce specific cellular immunity provides the experimental basis for breast cancer immunotherapy using genetically modified cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2008年第12期2146-2149,共4页
Journal of Southern Medical University
基金
中美合作研究基金(AN20020621)
广东省医学科研基金(A2005405)
关键词
乳腺癌
BA46
腺相关病毒
特异细胞免疫
breast cancer
BA46
adeno-associated virus
specific cellular immunity
