Kindlin-2在软骨细胞与间充质干细胞共培养中的调控作用及构建高质量组织工程软骨的应用研究

At present, there are still many difficulties remaining to ravel out in cartilage tissue engineering, especially in finding high quality and convenient sources of seeding cells. In the previous researches, the applicant has demonstrated that mesenchymal stem cells (MSCs) could stimulate and support chondrocytes to express more chondrocyte specific proteins in co-culture system and utilizing co-culture of chondrocytes with MSCs could construct tissue-engineered cartilage in higher quality. Our pilot experiment has showed that integrin-mediated signaling pathways could be activated by cell-cell contact in chondrocytes during co-culture with MSCs, and Kindlin-2 may play an important role in regulating this co-culture system. However, the specific mechanism remains unclear. Based on our pilot experiment, we hypothesize that in co-culture system the extracellular matrix formation and quality of tissue-engineered cartilage could be enhanced by two ways mentioned below. ①The cell-cell contact could activating "Integrin/Kindlin-2/ERK/SOX9" signaling pathway in chondrocytes; ②meanwhile, Kindlin-2 could bind with other transcription regular factors after making its way into the nucleus, and then directly regulate the expression of SOX9 and TβRI, which is a positive feedback to activate TGF-β/Smad/SOX9 signaling pathway. This project will deeply reveal the role of Kindlin-2 in such regulation mechanism in co-culture system from cell, tissue-engineered cartilage and animal model, and then estimate whether Kindlin-2 over-expressed chondrocytes could be an alternative seeding-cell source in cartilage tissue engineering.

优化种子细胞的来源和质量仍是软骨组织工程的研究难点。我们已证实与间充质干细胞（MSCs）共培养可促进软骨细胞合成II型胶原等特异性基质，以此构建的组织工程软骨质量更好。预实验结果表明细胞间直接接触引起整合素（integrin）信号通路激活是MSCs促进软骨细胞功能的重要途径之一，且Kindlin-2是其中的关键调控因子，但具体机制不明。我们推测其可能的调控机制为:①MSCs通过细胞间直接接触激活软骨细胞中Integrin/Kindlin-2/ERK/SOX9信号通路；②Kindlin-2入核与其他转录调控因子形成复合物直接调控SOX9和TβRI表达，正反馈激活Kindlin-2/TGF-β/Smad/SOX9信号通路。本研究将分别从细胞、组织工程软骨及动物水平验证上述Kindlin-2介导的调控机制，探讨使用Kindlin-2过表达的软骨细胞代替常规种子细胞构建高质量组织工程软骨的可能性。

关节软骨在外伤或炎症因子刺激下一旦发生损伤，常常难以进行有效的自身修复，最终发展为骨关节炎。近年来，随着组织工程技术和支架材料研究的不断进展，组织工程重建被认为是一种有前途的软骨损伤干预措施。特别需要关注的是，如何进一步优化种子细胞的来源和质量仍然是软骨组织工程研究的重点和难点之一。软骨细胞和间充质干细胞(MSCs)是目前常用的种子细胞，然而这两种细胞在用于构建组织工程软骨时均存在不足之处。研究人员尝试将这两种细胞系进行共培养，结果显示共培养可促进II型胶原（Col2）和聚集蛋白聚糖（Aggrecan）等软骨特异性细胞外基质成分的合成。然而，软骨细胞与MSCs共培养促进软骨特异性细胞外基质合成的具体调控机制尚不明确。本研究的结果表明：在软骨细胞与MSCs共培养系统中，Kindlin-2及其激活的PI3K/AKT信号能够促进软骨细胞分泌细胞外基质，并能抑制IL-1beta诱导的炎症。同时，体内实验表明软骨细胞与MSCs共培养能显著促进透明软骨再生。此外，进一步的体外研究结果表明：在直接接触共培养系统中与MSCs共培养的软骨细胞能够上调Kindlin-2表达，并激活PI3K/AKT信号通路，不仅能够增加Sox9和Col2表达，还可恢复线粒体膜电位并降低ROS水平和炎症条件下的细胞凋亡。该研究结果表明：软骨细胞与MSCs进行直接接触共培养可促进软骨形成，并确定Kindlin-2是该过程中的关键调节因子。本研究对软骨细胞与MSCs共培养的相互作用效应及调控机制进行探讨，并提出使用过表达Kindlin-2的软骨细胞代替传统的种子细胞，为进一步提高组织工程软骨质量提供了新思路和科学依据。