关节软骨损伤后自我修复能力极其有限，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；而经基因修饰的MSCs复合生物支架是目前修复软骨缺损（尤其是直径大于2cm）最有价值的方式。我们的前期研究发现，将Sox9单独或联合TGF-β基因导入MSCs后，其增殖、成软骨分化等显著提高。鉴于以上研究基础，本课题拟建立不同空隙等特性的壳聚糖3D生物支架，后将经重组腺相关病毒（rAAV）-Sox9基因修饰的含MSCs的骨髓穿刺液直接或经细胞培养后复合于上述3D支架，通过在体外培养和裸鼠体内、兔膝关节软骨缺损处移植，分析基因转染效率、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、软骨缺损修复效果等，明确最佳特性的壳聚糖支架以及与经sox9修饰的MSCs最佳组合，探索促进MSCs更安全稳定向透明软骨细胞分化的基因调控策略和软骨缺损修复方法。

Due to the intrinsic limited self-healing potential of articular cartilage, direct transplantation of genetically modified progenitor cells in bone marrow mesenchymal stem cells (MSCs) is an attractive strategy to conveniently facilitate the chondrogenic differentiation processes as a novel means to improve the intrinsic repair capacities of damaged articular cartilage in the local sites of injury; and moreover genetically modified MSCs composite scaffold is the most currently available and valuable strategy for the repair of cartilage defects (especially larger than 2cm). Our previous studies have demonstrated that independent overexpression of the pleiotropic growth factor TGF-β and IGF-I, and specialized chondrogenic transcription factor SOX9 via rAAV-mediated gene transfer upon both human and minipig bone marrow aspirates leaded to elevated levels of proliferation, matrix synthesis, and chondrogenic differentiation and meanwhile suppressed the hypertrophic and osteogenic differentiation processes over time. In light of the abovementioned research, this project intends to create a different novel characteristics of chitosan 3D scaffolds, incorporated with recombinant adeno-associated virus (rAAV) -Sox9 containing genetically modified MSCs in the bone marrow aspirates, either directly or after complex in cell culture 3D scaffold above, in vitro and in nude mice, and rabbit knee articular cartilage local defects, and subsequently, analysis of gene transfection efficiency, cell proliferation and apoptosis, differentiation into chondrocytes and extracellular matrix synthesis and cartilage hypertrophy and ossification, cartilage defect effects, to elucidate the best features and the best combination of chitosan scaffolds and MSCs modified by sox9 explore gene regulation approaches and promote cartilage defect repair.

关节软骨损伤后自我修复能力及其有限，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；而经基因修饰的MSCs复合生物支架是目前修复软骨缺损（尤其是直径大于2cm）最有价值的方式。我们已有的研究发现，通过人骨髓基质干细胞（hMSCs）的分离（bone marrow aspirates）和干细胞表面标记鉴定（FACS分选），建立了纯度较高的hMSCs单细胞克隆；并构建与鉴定、检测了人重组腺相关病毒(rAAV)-FLAG-hsox9质粒病毒载体的转染效率；获得经rAAV-FLAG-hsox9稳定转染人骨髓aspirates和hMSCs模型；经rAAV-FLAG-hsox9转染aspirates和hMSCs复合3D支架培养模型，通过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等明确了壳聚糖3D多孔支架最佳的hMSCs成软骨分化空隙大小约为100-200nm。最后，将经基因修饰的aspirates和hMSCs复合3D支架的裸鼠接种，通过比较植入前后aspirates和hMSCs复合3D支架颜色体积/重量的变化，采用免疫组化、RT-PCR分析并比较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等，证实了经rAAV-FLAG-hsox9转染的aspirates和hMSCs复合3D支架能稳定促进两者向软骨细胞样细胞分化，其成软骨关键靶基因表达明显升高，且组织相容性良好，未见明显血管增生等，为探索促进MSCs更安全稳定向透明软骨细胞分化的基因调控策略和软骨缺损修复提供了理论与实验依据。