整合素β1聚集取向和构象转变介导Wnt通路对脂肪间充质干细胞来源软骨样细胞表型"失稳"的调控机制研究

It is difficult for injured cartilage to undergo self-repair due to the lack of blood supply. Stem cell-derived chondroid cells currently play important role in tissue engineering based cartilage-repaired, however, undesirable phenotype "istability" caused by dedifferentiation often occur which greatly affect the application in tissue engineering based cartilage-repaired, though the underlying mechanism remains unclear. Studies have shown that integrin β1 mediates intracellular signal transduction pathway in cellular functions regulation through modulating aggregation status and conformational changes. We recent study found that knocked down of integrin β1 inhibit chondroid cells differentiation from stem cell as well as activate Wnt signal pathway, while ectopic expression of integrin β1 enhance the differentiation, which is consistent with the switch off of Wnt signal pathway. From the observation, we infer that integrin β1 regulate Wnt signal pathway through modulating aggregation status and comfomatinal changes, and evently affect chondroid cells differentiation. To demonstrate this, we will establish stable expression or knocked-down system for integrin β1, and then dynamic evaluating the aggregative state and comformational change in the process of cell differentiation by atomic force microscope and circular dichroism. To understand the potential mechanisms in terms of interactive protein between integrin β1 and canonical Wnt signal pathway, GST-pull down and Co-immunoprecipitation are used to validate the interaction-protein and potential target. In addition, composite polymer/quid crystal matrix elastic model is used as biological scaffold to further validate if the same mechanism is important to chondroid cells differentiation and cartilage development in vivo. Overall, this study will provide new insight into maintaining the phenotypes of chondroid cells, which in turn benefiting the application of tissue-engineered hyaline cartilage into clinical setting.

干细胞来源软骨样细胞去分化所致表型"失稳"严重影响关节软骨缺损修复效果，然而去分化机制仍不明确。研究证明，整合素β1通过聚集取向和构象转变介导胞内信号转导影响细胞的功能。我们近期研究发现，沉默干细胞整合素β1基因，经典Wnt通路持续开放，细胞丧失软骨向分化潜能；过表达该基因，经典Wnt通路持续关闭，细胞高效分化。由此推测整合素β1通过聚集取向和构象转变调节经典Wnt通路影响干细胞软骨向分化。研究拟通过体外干预脂肪间充质干细胞整合素β1表达，采用原子力显微镜针尖修饰、圆二色谱、GST-pull down和免疫共沉淀等技术证明之，应用聚合物/液晶动物模型体内观察整合素β1调控组织工程化软骨构建效果，从而探讨整合素β1聚集取向和构象转变影响经典Wnt信号转导，参与对干细胞软骨向分化调节的机制。结果将有助于高效稳定维持干细胞来源软骨样细胞表型，并为阐明细胞去分化机制及提高软骨修复质量提供理论基础。

干细胞来源软骨样细胞去分化所致表型“失稳”严重影响关节软骨缺损修复效果，然而去分化机制仍不明确。我们应用原子力显微镜探针修饰技术，在细胞培养的二维水平进行人脂肪间充质干细胞成软骨分化细胞形貌观察，发现长纺锤体细胞变成多边形细胞，长宽比减小，粗糙度增大。通过Integrin β1修饰的针尖与细胞膜表面跨膜蛋白受体的拉断力分析，发现整合素β1配体与受体结合概率进行性增加。研究表明分化过程中整合素β1聚集取向发生改变。我们通过高通量测序筛选过表达Integrin β1后表达有差异的基因，基于KEGG Pathway分析结果显示，在Cytokine-cytokine receptor interaction、PI3K-Akt signaling pathway、Rheumatoid arthritis、Pathways in cancer中富集到较多Integrin β1调控的基因，这提示Integrin β1可能通过细胞PI3K-Akt、Wnt通路影响细胞功能。进一步研究干细胞成软骨分化过程中，整合素β1与COL II、SOX9、GSK-3β和β-catenin同步增加。通过外源添加不同浓度梯度的Integrin β1诱导ADSCs分化，发现20ng/ml Integrin β1最佳，COL II、aggrecan、SOX9经Integrin β1诱导表达上调，纤维软骨标记物COL I没有变化。研究发现提示我们可以通过持续给予ADSCs 20ng/ml Integrin β1调控经典wnt通路进行透明软骨细胞诱导分化并维持表型不出现退变。糖尿病性骨关节炎患者关节液中的MMP-8和MMP-9两种炎症因子明显比非糖尿病性骨关节炎患者和正常人高，外周血单个核细胞脂多糖刺激后高反应分泌MMP-8，-9。由此推测糖尿病患者关节液高表达MMP-8，-9是糖尿病性骨关节炎发生的基础和重要原因，MMP-8，-9高表达可能作为负调控因素参与软骨退变。研究结果阐明了细胞去分化机制，有助于构建高效稳定的干细胞来源组织工程化软骨。