Wübbenhorst Daniela, Dumler Katja, Wagner Bettina, Wexel Gabriele, Imhoff Andreas, Gansbacher Bernd, Vogt Stephan, Anton Martina
Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
Arthritis Rheum. 2010 Jul;62(7):2037-46. doi: 10.1002/art.27461.
Treatment of cartilage defects is still challenging, primarily because of the poor self-healing capacity of articular cartilage. Gene therapy approaches have gained considerable attention, but, depending on the vector system used, they can lead to either limited or unrestrained gene expression, and therefore regulation of gene expression is necessary. This study was undertaken to construct an efficient tetracycline (Tet)-regulated, lentivirally mediated system for the expression of growth factor bone morphogenetic protein 2 (BMP-2) in primary rabbit chondrocytes that will allow for the induction and termination of growth factor gene expression once cartilage regeneration is complete.
Chondrogenic ATDC5 cells and primary rabbit chondrocytes were lentivirally transduced with different tetracycline-on (Tet-On)-regulated, self-inactivating vectors for the induction of expression of enhanced green fluorescent protein (eGFP) or BMP-2, using either a 1-vector system or a 2-vector system.
Expression of eGFP was induced on ATDC5 cells and chondrocytes. The highest induction rate and highest level of gene expression were reached when the spleen focus-forming virus long terminal repeat promoter was used to drive the reverse transactivator expression, after the addition of doxycycline, in chondrocytes. An up to 20-fold induction of Tet-mediated BMP-2 expression was observed on ATDC5 cells. The extent of induction and expression level of BMP-2 in chondrocytes were similar between the 1-vector system- and 2-vector system-infected cells (mean +/- SD 15.5 +/- 1.1 ng/ml and 14.6 +/- 0.4 ng/ml, respectively). In addition, prolonged induction and switching-off of BMP-2 expression, as well as repeated induction, were demonstrated. Production of proteoglycans, as shown by Alcian blue staining, demonstrated the functionality of the lentivirally expressed BMP-2 under induced conditions.
The lentivirally mediated Tet-On system is an effective strategy for efficient, repeatedly inducible expression of BMP-2 in primary rabbit chondrocytes. Therefore, use of this system in in vivo experiments may be a promising approach as a treatment strategy for cartilage defects.
软骨缺损的治疗仍然具有挑战性，主要原因是关节软骨的自我修复能力较差。基因治疗方法已受到广泛关注，但根据所使用的载体系统不同，可能导致基因表达受限或不受控制，因此基因表达的调控是必要的。本研究旨在构建一种高效的四环素（Tet）调控的慢病毒介导系统，用于在原代兔软骨细胞中表达生长因子骨形态发生蛋白2（BMP-2），以便在软骨再生完成后能够诱导和终止生长因子基因的表达。
使用1载体系统或2载体系统，用不同的四环素诱导（Tet-On）调控的自失活载体对软骨形成的ATDC5细胞和原代兔软骨细胞进行慢病毒转导，以诱导增强绿色荧光蛋白（eGFP）或BMP-2的表达。
在ATDC5细胞和软骨细胞中诱导了eGFP的表达。在软骨细胞中添加强力霉素后，当使用脾脏灶性形成病毒长末端重复启动子驱动反向反式激活因子表达时，达到了最高的诱导率和最高的基因表达水平。在ATDC5细胞上观察到Tet介导的BMP-2表达有高达20倍的诱导。1载体系统和2载体系统感染的细胞中，软骨细胞中BMP-2的诱导程度和表达水平相似（分别为平均±标准差15.5±1.1 ng/ml和14.6±0.4 ng/ml）。此外，还证明了BMP-2表达的延长诱导和关闭以及重复诱导。阿尔辛蓝染色显示蛋白聚糖的产生，证明了慢病毒表达的BMP-2在诱导条件下的功能。
慢病毒介导的Tet-On系统是在原代兔软骨细胞中高效、反复诱导表达BMP-2的有效策略。因此，在体内实验中使用该系统可能是一种有前景的软骨缺损治疗策略。
