Regeneration of a welt-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells
Regeneration of a welt-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells
复制标题
DOI：
10.1002/jgm.570
复制
发表时间：
2004-08-01
影响因子：
3.5
通讯作者：
Puchelle, E
中科院分区：
文献类型：
--
作者：
Castillon, N;Avril-Delplanque, A;Puchelle, E
背景 气道上皮损伤后，功能性上皮的快速再生对于恢复上皮屏障的完整性是必要的。从气道基因/细胞治疗的角度来看，我们分析了培养为三维（3-D）球状结构的人气道上皮细胞通过假型慢病毒载体长期有效转导的能力。还分析了 3-D 球状结构重新填充裸露气管基底膜和再生分化良好的气道上皮的能力。方法使用编码增强型绿色荧光蛋白 (eGFP) 的 HIV-1 衍生 VSV-G 假型慢病毒载体。从成熟的人胎儿气管和气道异种移植物中分离出气道上皮细胞，培养为 3-D 球状结构，并以 10 和 100 感染复数 (MOI) 进行转导，或在离体和体内模型中进行测定，以评估其再生能力。 结果 使用转导的分离胎儿气道上皮细胞对 SCID-hu 小鼠进行体内再增殖测定，结果表明慢病毒转导不会改变气道重构。 3-D 球体结构的转导显示，12% 的细胞在长达 80 天的时间内呈 eGFP 阳性。在离体和体内试验（NUDE-hu 小鼠）中，3-D 球状结构能够重新填充裸露的基底膜并重建分化良好的人气道表面上皮。结论 3-D 球状结构的有效和长期慢病毒转导及其再生分化良好的粘膜纤毛上皮的能力证明了这些 3-D 结构在人气道中的潜在相关性基因/细胞治疗。版权所有 (C) 2004 John Wiley Sons, Ltd.
Background Following injury to the airway epithelium, rapid regeneration of a functional epithelium is necessary in order to restore the epithelial barrier integrity. In the perspective of airway gene/cell therapy, we analyzed the capacity of human airway epithelial cells cultured as three-dimensional (3-D) spheroid structures to be efficiently transduced on long term by a pseudotyped lentiviral vector. The capacity of the 3-D spheroid structures to repopulate a denuded tracheal basement membrane and regenerate a, well-differentiated airway epithelium was also analyzed.Methods An HIV-1-derived VSV-G pseudotyped lentiviral vector encoding the enhanced green fluorescent protein (eGFP) was used. Airway epithelial cells were isolated from mature human fetal tracheas and airway xenografts, cultured as 3-D spheroid structures, and either transduced at multiplicity of infection (MOI) 10 and 100 or assayed in an ex vivo and in vivo model to evaluate their regeneration capacity.Results An in vivo repopulation assay in SCID-hu mice with transduced isolated fetal airway epithelial cells shows that lentiviral transduction does not alter the airway reconstitution. Transduction of the 3-D spheroid structures shows that 12% of cells were eGFP-positive for up to 80 days. In ex vivo and in vivo assays (NUDE-hu mice), the 3-D spheroid structures are able to repopulate denuded basement membrane and reconstitute a well-differentiated human airway surface epithelium.Conclusions The efficient and long-term lentiviral transduction of 3-D spheroid structures together with their capacity to regenerate a well-differentiated mucociliary epithelium demonstrate the potential relevance of these 3-D structures in human airway gene/cell therapy. Copyright (C) 2004 John Wiley Sons, Ltd.