单疱疹病毒结构蛋白BVP22的亚细胞定位及在细胞间的穿梭

Cellular localization and intercellular trafficking of bovine herpersvirus structural protein BVP22

背景与目的单疱疹病毒结构蛋白BVP22具有在细胞间穿梭的特性,并能够携带与其融合的蛋白从细胞内合成后穿梭到周围的细胞,克服了基因治疗中转染效率低的问题。本研究的目的是检测BVP22蛋白在细胞中的定位及体内外的穿梭,为将其应用于肺癌的基因治疗提供依据。方法Lipofectamin介导质粒pEYFP和pEYFPBVP22转染肺癌细胞系801D,G418筛选,建立单克隆细胞系。荧光观察确定BVP22的表达及定位。单克隆细胞系与母系801D以一定比例混合培养,采用流式细胞分选计数、免疫细胞化学染色检测BVP22在细胞间的穿梭。建立裸鼠移植瘤,脂质体包裹质粒注入瘤体检测BVP22在实体瘤中的穿梭。结果成功获得单克隆细胞系pEYFP801D和pEYFPBVP22801D。BVP22在细胞中的定位表现出多相性,多数为胞核定位,少数为胞质丝状定位。流式细胞计数显示,随培养时间延长,YFP阳性细胞的比例无明显增加。免疫细胞化学染色显示pEYFPBVP22801D与801D混合培养24h后,几乎所有细胞的胞核都染成了棕黄色;裸鼠移植瘤的免疫组化染色显示注射pEYFPBVP22的肿瘤组织大部分被染成棕黄色。结论BVP22主要定位于细胞核中,其胞核定位可能与细胞分裂及其在细胞间穿梭相关。在体内外BVP22均能够介导与其融合的蛋白在细胞间发生穿梭。

Background and objective The bovine herpersvirus structural protein BVP22 exhibits the remarkable property of intercellular trafficking whereby the protein fused to BVP22 spreads from the cell in which it is synthesized to surrounding cells. This function of BVP22 might be exploited to overcome the low efficiency of genes and gene products delivery, which is a major hurdle in gene therapy. The aim of this study is to investigate the cellular localization and intercellular trafficking of BVP22 in vitro and in vivo and provide scientific data for its application in gene therapy of human lung cancer. Methods 80ID cells were transfected respectively with plasmids pEYFP and pEYFP-BVP22 mediated by Lipofectamin and were selected by G418 to establish clone cell lines. The expression and cellular localization of BVP22 were examined by direct observation of YFP. Intercellular trafficking of BVP22 in vitro was detected by fluorescence-activated cell sorting （FACS） and immunocytochemical staining with YFP-antibody. Subcutaneous 801D tumors in nude mice were established to investigate the intercellular trafficking of BVP22 in vivo. Results Clone cell lines pEYFP-801D and pEYFP-BVP22-801D were established successfully. Cellular localization of BVP22 displayed heterogenicity. BVP22 was present in nuclei in most cells and only a few cells showed filamentous cytoplasm pattern. The results of FACS showed that the ratios of YFP-positive cells in mixed cells did not enhanced significantly. Im- munocytochemical staining demonstrated that the nuclei of almost all cells were stained positively after pEYFP-BVP22-801D and 801D were cultured for 24 h. Intercellular trafficking of YFP-BVP22 could be observed in subcutaneous 801D tumors in nude mice by immunohistochemical staining. Conclusion BVP22 displays nuclear localization in most cells. Its nuclear localization might be related to cell mitosis and intercellular trafficking. BVP22 can mediate intercellular trafficking of fusion protein in vitro and in vivo.