ADAMTS-4/5和Aggrecan基因改造的间充质干细胞和软骨细胞在软骨组织工程上的应用研究

关节软骨细胞外基质的降解是导致退行性骨关节炎的主要原因。关节软骨细胞外基质主要由Ⅱ型胶原和蛋白聚糖 (aggrecan) 组成，ADAMTS-4/5对蛋白聚糖的降解被认为在骨关节炎疾病发生早期发挥着关键作用。自体软骨细胞移植是再生医学中修复软骨损伤最好的方法之一，但是面临一些问题，如会造成供区继发软骨缺损，远期疗效不确定等。iPSCs技术为软骨组织工程种子细胞开辟了全新的来源途径，本课题拟利用TALEN靶向基因技术，对人尿液细胞来源的iPS细胞进行ADAMTS-4/5基因的敲除或aggrecan核心蛋白球间区Glu373-A1a374位点的基因突变，再从基因修饰的iPS细胞体外定向分化为间充质干细胞或软骨细胞，构建基因修饰的软骨组织，研究其对软骨缺损的修复。这种基因修饰可能更有利于维持软骨移植物细胞外基质的稳定，获得很好的远期疗效，为软骨损伤的修复提供更有效的治疗方法。

Chronic articular cartilage defects are one of the most common disabling conditions of elder people. Cartilage consists of a relatively small number of chondrocytes and abundant extracellular matrix (ECM) components. The major constituents of ECM are type II collagen and aggrecan. Chondrocytes synthesize and catabolize ECM macromolecules, while the matrix in turn functions to maintain the homeostasis of the cellular environment and the structure of cartilage. The primary cause of cartilage destruction is thought to be an elevation in the activities of proteolytic enzymes. The digestion of aggrecan by aggrecanases (ADAMTS-4/5) is considered a critical early event of acquired cartilage defects and results in arthritis. Today, autologous chondrocyte implantation (ACI) is the treatment of choice for severe cartilage defects, but this procedure suffers from several drawbacks. These include that the patient has to undergo multiple surgeries, and that in vitro expansion of chondrocytes is slow and dedifferentiation processes occur, which reduce the ability of cells to produce extracellular matrix. Transplantation of differentiated mesenchymal stem cells (a type of adult stem cell that can be transformed into chondrocyte-like cells) remains a promising approach for regenerative therapies, but often the tissue generated in vivo shows lower matrix accumulation and subsequent less mechanical stability as compared to chondrocyte-based transplantation. Induced pluripotent stem cells (iPSCs) have the capacity to overcome limitations associated with the current cell sources, primarily because large numbers of patient-matched cells with chondrogenic potential can be derived from a minimally invasively isolated starting cell population. As somatic cells that have been genetically reprogrammed to a pluripotent state, iPSCs demonstrate significant expansion potential while maintaining their multilineage differentiation capacity. In this study, we will produce integration-free human iPSCs that bear a knockout of the ADAMTS-4/5 gene or a knock-in modification of the aggrecan gene that produces a protein that cannot be digested by ADAMTS-4/5 at the critical cutting site. For this purpose we will use TALEN nucleases. These engineered iPSCs will be differentiated into MSCs (and then further into chondrocytes) or directly into chondrocytes and transplanted into animal models of cartilage damage. We anticipate that this approach will favorably alter the balance between synthesis and degradation of the ECM in diseased joints, thus allowing more efficient recovery. Our proposal is highly relevant because if successful, a similar approach may be suitable for patients with cartilage problems in the near future, which would consequently improve the quality of life of millions of people in China and also elsewhere.