Her2/neu基因重组腺相关病毒转染人外周血来源树突状细胞的免疫功能

Immunostimulatory effect of human peripheral blood dendritic cells transfected by adeno- associated virus containing Her2/neu gene

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(DC),并检测其免疫功能。方法采用Ficoll分离健康人外周血中的单个核细胞。将其分成两组,给其中一组加入病毒。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,白细胞介-4、肿瘤坏死因子-α培养。第7天收获成熟DC。流式细胞仪检测DC表型。3H-TdR检测同种混合淋巴细胞反应。MTT法检测DC诱导T细胞的杀伤活性。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 observe the immunostimulatory effect of human peripheral blood dendritic cells （DCs） transfected by Her2/neu gene delivered by adeno-associated virus. Methods The mononuclear cells in healthy donors were isolated by Ficol-Hypaque density gradient separation and divided into transfection group and control group without transfection by the recombinant virus. The cells were initially cultured in RPMI 1640 medium supplemented with 10% AB human serum, followed by addition of granulocyte-macrophage colony-stimulating factor （GM-CSF）, interleukin （IL）-4 and tumor necrosis factor （TNF）-α into the medium. The surface markers of DC were detected by flow cytometry, allogeneic mixed lymphocyte reaction assayed by ^3H-TdR incorporation and the specific killing activity of T cells evaluated by MTT assay. Results The expression rates of CDla, CD86 and CD83 of the transfected DCs were 98.10%, 99.42%, and 84.59%, and those of the non-transfected DCs were 92.69%, 98.07%, and 82.72%, respectively, showing no obvious differences between the two groups. The rates of CD4 and CD80 expression were 61.02% and 97.61%, respectively, in the transfected DCs, significantly higher than those in the non-transfected cells （36.19% and 55.5%）. Both groups of DCs stimulated a strong T cell proferation response. The transfected DCs were capable of inducing specific killing of the target tumor cells, with the highest killing rate of （39.7±7.2）%. Conclusion The immunostimulatory effect of human peripheral blood DCs can be enhanced by Her2/neu gene transfection mediated by adeno-associated virus.