Gelse K, Jiang Q J, Aigner T, Ritter T, Wagner K, Pöschl E, von der Mark K, Schneider H
Department of Experimental Medicine I, University of Erlangen-Nuremberg, Erlangen, Germany.
Arthritis Rheum. 2001 Aug;44(8):1943-53. doi: 10.1002/1529-0131(200108)44:8<1943::AID-ART332>3.0.CO;2-Z.
To assess the advantages and disadvantages of a direct adenoviral and a cell-mediated approach to the induction of cartilage formation in joints by transfer of growth factor genes.
Adenoviral vectors carrying insulin-like growth factor 1 (IGF-1) or bone morphogenetic protein 2 (BMP-2) complementary DNA were constructed and applied to primary human and murine chondrocytes or fibroblasts. Transgene expression was quantified by enzyme-linked immunosorbent assay. Direct injection of these vectors or AdLacZ, a reporter gene vector, into mouse knee joints was compared with the transplantation of syngeneic fibroblasts (infected ex vivo with the same vectors) with respect to virus spread, immune response, and cartilage formation by use of histologic, immunohistochemical, and molecular analyses.
AdIGF-1 and AdBMP-2 efficiently infected all cell types tested. Human cells secreted biologically relevant levels of protein over a period of at least 28 days. Direct transfer of AdLacZ into mouse knee joints resulted in positively stained synovial tissues, whereas AdLacZ-infected fibroblasts settled on the surface of the synovial membranes. Inadvertent spread of vector DNA into the liver, lung, and spleen was identified by nested polymerase chain reaction in all mice that had received the vector directly; this rarely occurred following fibroblast-mediated gene transfer. Direct injection of AdBMP-2 induced the synthesis of new cartilage in periarticular mesenchyme, accompanied by extensive osteophyte formation. When AdBMP-2 was administered by injecting ex vivo-infected fibroblasts, cartilage formation was observed only in regions near the injected cells. AdIGF-1 treatment did not lead to morphologic changes. Importantly, fibroblast-mediated gene transfer avoided the strong immune response to adenovirus that was elicited following direct application of the vector.
Our results indicate that cell-mediated gene transfer provides sufficient BMP-2 levels in the joint to induce cartilage formation while avoiding inadvertent vector spread and immune reactions.
通过生长因子基因转移，评估直接腺病毒法和细胞介导法在关节诱导软骨形成方面的优缺点。
构建携带胰岛素样生长因子1（IGF-1）或骨形态发生蛋白2（BMP-2）互补DNA的腺病毒载体，并应用于原代人及小鼠软骨细胞或成纤维细胞。通过酶联免疫吸附测定法定量转基因表达。将这些载体或报告基因载体AdLacZ直接注射到小鼠膝关节中，并与同基因成纤维细胞（离体用相同载体感染）移植在病毒传播、免疫反应和软骨形成方面进行比较，采用组织学、免疫组织化学和分子分析方法。
AdIGF-1和AdBMP-2能有效感染所有测试的细胞类型。人细胞在至少28天的时间内分泌生物学相关水平的蛋白质。将AdLacZ直接转移到小鼠膝关节中导致滑膜组织呈阳性染色，而AdLacZ感染的成纤维细胞定居在滑膜表面。通过巢式聚合酶链反应在所有直接接受载体的小鼠中鉴定出载体DNA意外扩散到肝脏、肺和脾脏；而成纤维细胞介导的基因转移很少发生这种情况。直接注射AdBMP-2可诱导关节周围间充质中合成新的软骨，并伴有广泛的骨赘形成。当通过注射离体感染的成纤维细胞给予AdBMP-2时，仅在注射细胞附近的区域观察到软骨形成。AdIGF-1处理未导致形态学改变。重要的是，成纤维细胞介导的基因转移避免了直接应用载体后引发的对腺病毒的强烈免疫反应。
我们的结果表明，细胞介导的基因转移在关节中提供了足够的BMP-2水平以诱导软骨形成，同时避免了载体的意外扩散和免疫反应。
