关节软骨损伤后自我修复能力非常有限，经基因修饰的间充质干细胞（MSCs）可稳定分化为软骨细胞，是治疗软骨缺损最有前景的种子细胞。我们的前期研究发现，Sox9单独或联合TGF-β基因过表达MSCs，其细胞增殖、成软骨分化和细胞外基质合成能力显著增强。鉴于Wnt/β-catenin通路也可直接调节MSCs成软骨分化，并结合我们前期成果，本课题拟分别构建MSCs 3D微聚和aspirates培养模型，采用重组腺相关病毒（rAAV）-RNAi特异性敲低β-catenin且同时过表达Sox9；在上述过程中加入Wnt3a和/或阻断剂CaMKⅡ，通过检测基因转染效率、细胞增殖和凋亡、成软骨分化和细胞外基质合成及软骨肥大化和骨化等指标，明确Wnt/β-catenin信号通路与sox9在MSCs成软骨细胞分化过程中的相互作用及分子机制，探索促进MSCs更佳、更安全和更稳定向透明软骨细胞分化的基因调控策略。

Articular cartilage has a limited potential for self-healing. Transplantation of genetically modified progenitor cells like bone marrow-derived mesenchymal stem cells (MSCs) is an attractive strategy to improve the intrinsic repair capacities of damaged articular cartilage. In the previous research, we found that prolonged TGF-β/sox9 co-overexpression was achieved in chondrogenically-induced hMSCs upon co-transduction via rAAV for up to 21 days, leading to enhanced proliferative, biosynthetic, and chondrogenic activities relative to control treatments, especially when co-applying the candidate vectors at the highest vector doses tested. Optimal co-administration of TGF-β with sox9 also advantageously reduced hypertrophic differentiation of the cells. As it has already been certified that Wnt/β-catenin pathway also exerts direct regulation on the process of hMSCs chondrogenic differentiation, we hypothesized that combined use of Wnt/β-catenin pathway and core transcriptional factor sox9 might promote hMSCs chondrogenic differentiation. In the present study, we will establish hMSCs 3D micromass (pellet) and bone marrow aspirates models, apply the recombinant adeno-associated virus (rAAV)-RNAi specific targeting β-catenin and simultaneously overexpression of sox9, and add Wnt3a and/or block CaMKⅡ in the above-mentioned procedure. Efficiency of gene transfection, cell proliferation and apoptosis, chondrogenic differentiation and extracellular matrix synthesis, cartilaginous hypertrophy and ossification will be analyzed. Finally, the findings will clearly demonstrate the possible interaction and molecular mechanisms between Wnt/β-catenin signaling pathway and sox9 in the process of hMSCs chondrogenic differentiation, and the possibility of modifying by combined therapeutic gene transfer as potent, safe and stable future strategies for implantation in clinically relevant animal models of cartilage defects in vivo.