You Chang-xuan, Su Jin, Liao Wang-jun, Zhang Jun-yi, Liu Yong, Paul L Hermonat, Luo Rong-cheng
Department of Oncology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2008 Dec;28(12):2146-9.
To study the feasibility of transfecting breast cancer BA46 gene into dendritic cells (DCs) using adeno-associated virus (AAV) to induce specific cellular immunity.
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-alpha(TNF-alpha). 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 (3)H-thymidine incorporation assay. The expressions of interferon-gamma (IFN-gamma), IL-4, CD4, CD8, CD25 and CD69 in the CTLs were analyzed with cytometry, and the cytotoxicity of the CTLs was evaluated with (51)Cr-release assay using BA46-positive breast cancer cell line Hs578T as the target.
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-gamma, and showed strong cytotoxicity against BA46-positive breast cancer cell line Hs578T, which was BA46 antigen-specific and MHC-limited.
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.
研究利用腺相关病毒（AAV）将乳腺癌BA46基因转染至树突状细胞（DCs）以诱导特异性细胞免疫的可行性。
通过密度梯度离心从健康供体的外周血中分离单核细胞（DC前体），用rAAV/BA46/Neo病毒原液感染（转染组）或用293细胞裂解物脉冲处理（对照组）。两组均用粒细胞-巨噬细胞集落刺激因子（GM-CSF）、白细胞介素-4（IL-4）和肿瘤坏死因子-α（TNF-α）诱导DC前体成熟。第7天，收集DCs并与T细胞按1:20的比例混合以诱导细胞毒性T淋巴细胞（CTL）。使用³H-胸腺嘧啶核苷掺入法评估DCs刺激T淋巴细胞增殖的能力。用流式细胞术分析CTL中干扰素-γ（IFN-γ）、IL-4、CD4、CD8、CD25和CD69的表达，并以BA46阳性乳腺癌细胞系Hs578T为靶细胞，用⁵¹Cr释放法评估CTL的细胞毒性。
转染BA46基因的DCs表现出强大的刺激T淋巴细胞增殖的能力。转染后的DCs诱导产生的CTL群体高表达CD8、CD69和IFN-γ，并对BA46阳性乳腺癌细胞系Hs578T表现出强烈的细胞毒性，具有BA46抗原特异性和MHC限制性。
BA46基因成功转染至DCs并诱导特异性细胞免疫，为利用基因修饰细胞进行乳腺癌免疫治疗提供了实验依据。
