摘要
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 study the feasbility of BA46 gene transferring dendritic cells used in therapy of breast cancer. Methods Drew the peripheral blood from the healthy donor, isolated the mononuclear cells（DC precursor） with density gradient centrifugation, cultured with AIM-V medium, washed out the suspending cells after 5 hours. The adherent cells were divided into 2 groups, one is gene transfer group, another is control group. To observed the morphologic features of DC with light microscope, analyzed the surface marker CDS0, CD86, CD40 with flow cytometry. Mixed the DCs and T cells at ratio of 1 to 20, cultured in AIM-V medium contain 5% human plasma, induced by IL-2 and GM-CSF, finally got cytotoxic T lymphocyte（CTL） after 7 days. Test the cytotoxicity of the CTL by ^51Cr releasing with BA46 positive breast cancer cell line Hs578T as the target and to analyze the ratio of CD8/CD4 and CD8/CD56 in CTL population with flow cytometry. Results The DCs transfected with BA46 gene expressed the higher level of surface marker CD80, CD86, CD40 than that of control group. The CTL induced by the gene transferring DCs obtained high cytotoxicity activity to the BA46 positive breast cancer cell line Hs578T which is BA46 antigen specificity and MHC limited. The CTL population also got much higher ratio of CD8/CD4 and CD8/CD56 than that of the control group（the lysate group）. Conclusion The success of BA46 gene transducing DCs and inducing CTL set up a base for breast cancer immunotherapy with DC gene transfer.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2005年第7期574-577,共4页
Chinese Journal of Microbiology and Immunology
基金
中美合作研究基金资助项目(编号AN20020621)
