Her2/neu基因重组腺相关病毒转染人外周血树突状细胞的免疫功能

Adeno-assoiciated virus-mediated Her2/neu gene transfection enhances the immunostimulatory capacity of human dendritic cells in vitro

目的Her2/neu基因重组腺相关病毒(rAAV-Her2/neu)转染人外周血树突状细胞(dendriticcell,DC),并检测其免疫功能。方法检测乳腺癌细胞MCF7的HLA基因分型。采用与乳腺癌细胞株SK-BR-3、MCF7的HLA基因分型一致的健康人外周血,Ficoll分离取得单个核细胞,各分两组,分别加入病毒和不加病毒。应用含10%人AB血清的RPMI-1640培养基及粒细胞-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)培养。第7天收获成熟DC。流式细胞仪检测DC表型。MTT法检测DC诱导T细胞的杀伤活性,此步骤重复4次取平均值。结果加病毒组CD1a、CD86和CD83分别为:98.10%、99.42%、84.59%;无病毒组为:92.69%、98.07%、82.72%,组间差别不明显。加病毒组CD40、CD80分别为:61.02%、97.61%;无病毒组为:36.19%、55.5%,加病毒组高于无病毒组。两组均能刺激T淋巴细胞增殖。加病毒组DC可诱导特异性杀伤,最高杀伤率(39.7±7.2)%。结论rAAV-Her2/neu转染的DC有更强的免疫功能。

Objective To evaluate the immunostimulatory capacity of human peripheral blood dendritic cells （DCs） with Her2/neu gene transfection mediated by adeno-assoiciated virus. Methods The HLA genotypes of the breast cancer cells SK-BR-3 and MCF7 were determined, and the mononuclear cells from healthy donors with matching HLA genotype were isolated by Ficoll-Hypaque density gradient separation. The isolated cells were divided into two groups with or without transfection with the recombinant virus harboring Her2/neu gene. The cells were cultured for 7 days in RPMI 1640 medium supplemented with 10% AB human serum, GM-CSF, interleukin-4 （IL-4） and tumor necrosis factor-el （TNF-α. The mature DCs were then harvested from the cell culture and their phenotypes were identified using flow cytometry. MTT assay was employed to examine the specific killing activity of the T cells induced by the DCs. Results The DCs transfected with the recombinant adeno-assoicated virus expressed CDla, CD86 and CD83 at the rate of 98.10%, 99.42%, and 84.59%, and those without the viral transfection expressed the markers at the rate 92.69%, 98.07%, and 82.72%, respectively, showing no obvious differences in the phenotypes of the two DCs. The transfected DCs, however, showed markedly higher expression rates of CD40 and CD80 than the non-transfected DCs （61.02% vs 36.19%, and 97.61% vs 55.5%, respectively）. The DCs, irrespective of the transfection, showed comparable capacities in stimulating T cell proliferation. The transfected DCs exhibited the capacity of inducing the T cells to specifically kill the target tumor cells, with the highest killing rate of （39.7± 7.2）%. Coclusion The immunostimulatory capacity of human peripheral blood DCs are enhanced by Her2/neu gene transfection mediated by adeno-assoiciated virus.