Light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes.
Light-activated gene transduction enhances adeno-associated virus vector-mediated gene expression in human articular chondrocytes.
复制标题
光激活基因转导增强人关节软骨细胞中腺相关病毒载体介导的基因表达。
DOI：
10.1002/art.10433
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发表时间：
2002
期刊：
影响因子：
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通讯作者：
Schwarz,EdwardM
中科院分区：
文献类型：
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作者：
Ulrich-Vinther,Michael;Maloney,MichaelD;Goater,JJeffrey;Soballe,Kjeld;Goldring,MaryB;O'Keefe,RegisJ;Schwarz,EdwardM
目的探讨紫外线（UV）作为佐剂对重组腺相关病毒（rAAV）转导人关节软骨细胞的影响。方法将原代关节软骨细胞和永生化软骨细胞（tsT/AC 62）暴露于不同剂量的UV（0 ~ 1，000 J/m2），并以不同感染复数（MOI）感染含增强型绿色荧光蛋白（EGFP）基因的rAAV。在感染后第2、4和8天，通过荧光激活细胞分选分析细胞的活力和EGFP表达。为了评估完整关节软骨的转导效率，将全层外植体暴露于紫外线（0-200 J/m2），感染rAAV-eGFP，并通过免疫组化分析转导。ResultsUV暴露的毒性在原代和永生化软骨细胞培养物中分别在≥500 J/m2和≥200 J/m2剂量下观察到。转导效率依赖于UV剂量、MOI和时间。在细胞系中，UV对转导细胞百分比的佐剂效应是适度的，但100 J/m2使转导细胞的平均荧光强度（MFI）增加4倍。相比之下，UV处理对原代软骨细胞的转导效率有显著影响，在100 J/m2的UV光和103 MOIs下暴露8天后，其达到100%左右。在相同条件下，200 J/m2的UV光使MFI增强7倍。在软骨外植体中，浅表、中间或基底区外植体边缘的转导软骨细胞数量没有差异;然而，200 J/m2的UV光在低MOI下使转导效率增加2倍。在外植体的中心，表面软骨细胞被有效地转导;在中间区和基底区的那些在任何条件下都不能被有效地转导。在表层软骨细胞中，低MOI和200 J/m2的UV光使转导效率增加3倍（至100%）。（不显著影响细胞活力）显著增强rAAV感染的原代软骨细胞培养物和完整关节软骨浅层软骨细胞中转导基因的转导效率和表达，软骨这些发现支持了这样的概念，即紫外线激活的基因转导可用作具有低病毒滴度的体内rAAV关节软骨基因治疗的佐剂，以预防和/或治疗关节炎。
ObjectiveTo evaluate the effects of ultraviolet (UV) light as an adjuvant for recombinant adeno‐associated virus (rAAV) transduction in human articular chondrocytes.MethodsPrimary articular chondrocytes and immortalized chondrocytes (tsT/AC62) were exposed to various doses of UV light (0–1,000 J/m2) and infected at various multiplicities of infection (MOIs) with rAAV containing the enhanced green fluorescent protein (EGFP) gene. Cells were analyzed for viability and EGFP expression by fluorescence‐activated cell sorting on days 2, 4, and 8 following infection. To evaluate the transduction efficiency in intact articular cartilage, full‐thickness explants were exposed to UV light (0–200 J/m2), infected with rAAV‐eGFP, and analyzed for transduction via immunohistochemistry.ResultsToxicity from UV exposure was observed at doses ≥500 J/m2and ≥200 J/m2in primary and immortalized chondrocyte cultures, respectively. Transduction efficiency was dependent on the UV dose, MOI, and time. In the cell line, the adjuvant effect of UV on the percentage of cells transduced was modest, but 100 J/m2increased the mean fluorescence intensity (MFI) of the transduced cells 4‐fold. In contrast, UV treatment had a profound effect on the transduction efficiency of primary chondrocytes, which reached ∼100% after exposure to 100 J/m2of UV light and 103MOIs for 8 days. Under the same conditions, 200 J/m2of UV light enhanced the MFI 7‐fold. In cartilage explants, there was no difference in the number of transduced chondrocytes at the edge of the explants in the superficial, intermediate, or basal zones; however, 200 J/m2of UV light increased the transduction efficiency 2‐fold at a low MOI. In the center of the explants, the superficial chondrocytes were efficiently transduced; those in the intermediate and basal zones could not be efficiently transduced under any condition. In the superficial chondrocytes, a low MOI and 200 J/m2of UV light increased the transduction efficiency 3‐fold (to 100%).ConclusionUV light at doses of up to 200 J/m2(which do not significantly affect cell viability) significantly enhances the transduction efficiency and expression of the transduced gene in cultures of rAAV‐infected primary chondrocytes and in chondrocytes in the superficial zone of intact articular cartilage. These findings support the concept that UV‐activated gene transduction could be used as an adjuvant for in vivo rAAV articular cartilage gene therapy with low viral titers to prevent and/or treat arthritis.