Wnt/β-catenin信号通路在sox9基因介导的hMSCs成软骨分化中的作用及机制研究

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.

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。

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（hMSCs）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。项目组成员已完成课题申请书的如下工作：①通过分离人骨髓基质干细胞（hMSCs）、干细胞克隆化培养，hMSCs表面标记鉴定（FACS分选），显示人骨髓基质干细胞（hMSCs）表达CD71+和CD105+，而CD34-，采用单个细胞干细胞分选克隆化培养技术进行培养，随后，构建hMSCs 3D微聚培养模型，并采用含1 ng/ml FGF-2软骨诱导培养基进行成软骨诱导分化培养及检测诱导效率；②与此同时，课题组成员利用在德国做博士后期间所学的基因治疗的方法及相关技术手段，构建了rAAV-β-catenin-RNAi + rAAV-FLAG-hsox9质粒载体，并进行了上述载体的鉴定以及病毒转染效率的检测，接近100%；③在上述两步顺利完成的基础上，课题组采用rAAV-β-catenin-RNAi+rAAV-FLAG-hsox9病毒载体单独或联合转染hMSCs 3D微聚培养或人骨髓aspirates培养模型，转染后通过ELISA定量、Hoechst 33258法及DMMB法、H&E, Safr. O和Toluidine blue染色、I型胶原和X型胶原、II型胶原和Aggrecan免疫组化染色、实时荧光定量RT-PCR、免疫组化染色和Western blot检测β-gal、β-catenin和sox9等来检测上述模型中病毒转染效率、Wnt信号通路及成软骨分化相关基因的mRNA及蛋白质水平的表达；其次，体内试验部分，将经基因修饰的人骨髓aspirates接种至裸鼠背部皮下，再次检测上述模型中病毒转染效率、Wnt信号通路及成软骨分化相关基因的mRNA及蛋白质水平的表达；发现rAAV-β-catenin-RNAi + rAAV-FLAG-hsox9能发挥协同促进hMSCs成软骨分化，且效率远大于两者单独加入培养体系中；④在敲低或正常β-catenin表达时，加入Wnt3a和/或阻断CaMKⅡ（KN93），观察sox9的成软骨作用，结果发现上述hMSCs成软骨分化过程部分被阻断。