Wisp3调控基质金属蛋白酶分泌参与软骨稳态平衡的机制与应用研究

项目名称： Wisp3调控基质金属蛋白酶分泌参与软骨稳态平衡的机制与应用研究

项目编号： No.30872617

项目类型： 面上项目

立项/批准年度： 2009

项目学科： 生物科学

项目作者： 周后德

作者单位： 中南大学

项目金额： 34万元

中文摘要： 传统理论认为软骨是永久性的静态组织，面对众多的软骨发育不良与关节退行性变一筹莫展。我们以一个珍贵的遗传性软骨发育不良家系为突破口，初步揭示了Wisp3突变造成的软骨基质和降解酶系的表达与分泌障碍是软骨发育不良的主因之一，发现软骨的稳态维持可能是软骨降解与形成的缓慢动态平衡过程。然后我们通过逆转录病毒基因转移系统，在正常软骨细胞和突变软骨细胞中显性负性突变、基因剔除和组成性激活Wisp3 表达，考察Wisp3 的表达分泌变化；比较了正常关节软骨细胞、骨关节炎患者软骨细胞及SEDT-PA患者软骨细胞在体外成软骨能力的差异，三种细胞增殖分化能力、分泌软骨基质、基质金属蛋白酶等的差异；建立了可用于软骨联合三维支架与沉淀培养技术；在新西兰白兔的关节内注射上述Wisp3 基因修饰的逆转录病毒，观察其关节软骨形态组织学、软骨基质与细胞微结构、软骨生物力学与分子生物学变化，遗憾的是这一结果显示Wisp3 基因修饰的逆转录病毒与对照安慰组差别并不明显，后续研究将进一步改善Wisp3 基因的修饰方式，增加Wisp3 基因表达产物在软骨组织中的稳定性。

中文关键词： 软骨；wisp3 基因；软骨转换；基质金属蛋白酶

英文摘要： The cartilage was generally thought to be a static tissue which can't beregenerated,little advance has been achieved during the past ten years in the cartilage repair for osteoarthritis and bone dysplasia. We studied the cellular and molecular responses in progressive pseudorheumatoid dysplasia articular cartilage associated with compound heterozygous wisp3 gene mutation, and found that the expression and secretion disorder of cartilage matrix and the proteinase for matrix degradation is one of the main culprit of the cartilage dysplasia, which indicated that the cartilage is not a static tissue, and all the components in cartilade maintain a relatively dynamic balance. We then knocked-out/down and over-expressed the wild type and mutant wisp3 gene in normal and wisp3 mutant (from the patient) articular chondrocytes by retrovirus gene transfer system, obeserved the secretion and function of wisp3 protein, and the binding ability of the secreted wisp3 protein to it's ligand or receptor. The difference of cellular proliferation and differentiation, extracellular matrix and MMP expression were observed in normal chondrocytes, osteoarthritis chondrocytes and SEDT-PA chondrocytes. Then compared the difference of the cartilage formation ability and the quality of cartilage formed in vitro by each kind of genetic modified chondrocytes by in vitro three dimensional culture.The New-Zealand white rabbits were intra-articular injected with retrovirus encoded different type of wisp-3 gene,the difference of the morphology of articular cartilage, cartilage matrix and microstructure,biomechanics and molecular biology in each groups of rabbits were analyzed, however, no diference was found in Wisp-3 retrovirus injection group and control group (injected with PBS), next we will further improve the wisp-3 retrovirus and hopefuly we can get the positive result.

英文关键词： Cartilage; Wisp3; Cartilage turnover; MMPs