Involvement of glycosaminoglycans in vesicular stomatitis virus G glycoprotein pseudotyped lentiviral vector-mediated gene transfer into airway epithelial cells
Involvement of glycosaminoglycans in vesicular stomatitis virus G glycoprotein pseudotyped lentiviral vector-mediated gene transfer into airway epithelial cells
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DOI：
10.1002/jgm.1248
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发表时间：
2008-12-01
影响因子：
3.5
通讯作者：
Conese, Massimo
中科院分区：
文献类型：
--
作者：
Copreni, Elena;Castellani, Stefano;Conese, Massimo
背景人类免疫缺陷病毒(HIV-1)慢病毒(LentiVirus，LV)介导的呼吸道上皮细胞转导过程中表面分子参与的研究尚未见报道。本研究旨在探讨糖胺多聚糖(GAG)在第三代水泡性口炎病毒G糖蛋白(VSV-G)假型LV载体介导的呼吸道极化上皮细胞体外基因转移中的作用。方法体外培养人支气管(16HBE-S1)和气管(CFT1-C2)上皮细胞，用IN载体多尿路(PPT)-绿色荧光蛋白(GFP)进行转导。用细胞荧光法和共聚焦显微镜检测紧密连接标志ZO-1和GAG的定位。结果在塑料贴壁细胞中进行了大量的实验参数优化(感染过程中有聚丙稀烯存在、有LV颗粒存在的孵育时间、感染与基因表达分析相交的时间)。聚丁二烯具有细胞毒性，不再继续使用。在CFT1-C2极化细胞中，EGTA处理后跨上皮阻力降低20%，紧密连接部位ZO-1定位减少，GFP阳性细胞增加31%。硫酸乙酰肝素均匀分布于细胞表面。肝素和可溶性硫酸软骨素A和B以剂量依赖的方式抑制LV介导的转导。结论GAG参与了VSV-G在呼吸道上皮细胞的转导过程。版权所有(C)2008 John Wiley&Sons，Ltd.
Background The involvement of surface molecules in HIV-1-derived lentivirus (LV)-mediated transduction of airway epithelial cells has not been studied so far. The present study aimed to evaluate the role of glycosaminoglycans (GAGS) in gene transfer mediated by a third generation vesicular stomatitis virus G glycoprotein (VSV-G) pseudotyped LV vector in an in vitro model of polarized airway epithelial cells.Methods Human bronchial (16HBE-S1) and tracheal (CFT1-C2) epithelial cells were grown either on plastic or on filters and transduced with the IN vector polypurine tract (PPT)-green fluoresecent protein (GFP). Zonula Occludens (ZO)-1, a marker of tight junction, and GAG localization were assessed by cytofluorimetry and confocal microscopy. Soluble GAGS and removal of cell surface GAGS were used to affect LV-mediated transduction.Results Extensive optimization of experimental parameters (presence of polybrene during the infection, the incubation time in the presence of LV particles, period of time intercurring between infection and gene expression analysis) was carried out in plastic-adherent cells. Polybrene resulted to be cytotoxic and was not further used. In CFT1-C2 polarized cells, EGTA treatment determined a 20% decrease in transepithelial resistance, a diminished ZO-1 localization at the tight junction location and a 31% increase in GFP positive cells. Heparane sulfate was distributed evenly on the cell surface. Heparin and soluble chondroitin sulfate A and B inhibited LV-mediated transduction in a dose-dependent fashion. These results were confirmed upon enzymatic removal of GAGS from the cell surface.Conclusions Taken together, these results show that GAGS are involved in VSV-G IN transduction of airway epithelial cells. Copyright (C) 2008 John Wiley & Sons, Ltd.