E-钙粘蛋白在三维球形体培养维持MSCs干性中的作用机制研究

The stemness properties of stem cells may govern their therapeutic potentials in clinical applications. Mesenchymal stem cells (MSCs) represent a valuable source of stem cells for regenerative medicine, but the loss of their stemness during in vitro expansion remains a major roadblock. Previous studies have indicated that the three-dimensional (3D) culture have more efficient on the stemness maintenance of MSCs compared with the two-dimensional culture. However, the mechanisms that enhance the stemness properties of MSCs in 3D culture are intriguing but unclear.. Several studies have demonstrated that spheroid formation of MSCs can maintain their stemness properties, which is consistent with our previous studies. In our unpublished data, it has been showed that the increased expression of E-cadherin was detected in MSC spheroids, and their cloning efficiency was improved. Moreover, it has been reported that the E-cadherin in embryonic stem cells plays an important role in maintaining stemness properties and is also involved in restoring their pluripotency. Therefore, we hypothesize that E-cadherin involved in the stemness maintenance of mesenchymal stem cells cultured in 3D spheroid via their catenins. . For this purpose, 3D spheroid will be used as a 3D culture model to incubating adipose tissue derived stem cells. The effects of E-cadherin, catenins, target genes of catenins and feedback for E-cadherin on stemness properties of MSCs will be investigated through gene interference, co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP) and reverse ChIP. In all, the mechanisms by which E-cadherin mediated the stemness maintenance of MSCs under the 3D spheroid will be elucidated, which will contribute to the clinical application of MSC-based therapy.

间充质干细胞(MSCs)在体外扩增培养过程中逐渐失去干性是其临床转化面临的瓶颈问题。三维培养较二维培养能更好维持干细胞干性，前期我们发现三维球形体培养能有效维持MSCs干性，但其机制尚不明确。预实验证实E-钙粘蛋白在MSCs球形体内表达升高并提高其克隆形成率，根据E-钙粘蛋白在胚胎干细胞干性维持中的关键作用，我们提出假设：E-钙粘蛋白经其胞内连环蛋白调控三维球形体培养MSCs的干性维持。本项目拟以生物反应器内球形体培养MSCs为研究对象，首先观察E-钙粘蛋白在球形体培养维持MSCs干性中的作用；其次评价胞内连环蛋白对MSCs干性的影响并探讨其分子机制；最后关注靶基因对E-钙粘蛋白的反馈调节作用，以阐明E-钙粘蛋白在三维球形体培养维持MSCs干性中的作用机制，力图从三维立体的新视角阐述MSCs干性维持的作用机制，为探索能有效维持干细胞干性的大量扩增培养方法提供理论依据，促进干细胞的临床转化。

干细胞的干性决定了其临床治疗潜能。间充质干细胞(MSCs)是再生医学重要的干细胞来源，但在体外扩增培养过程中逐渐失去干性是临床转化面临的瓶颈问题。三维培养较二维培养能更好维持MSCs干性，但其机制尚不明确。我们以往研究表明三维球形体培养有效维持MSCs干性，E-钙粘蛋白在MSCs球形体中的表达显著升高。E-钙粘蛋白在胚胎干细胞的干性中发挥重要作用。因此，我们推测E-钙粘蛋白在球形体培养MSCs的干性维持中发挥关键作用。.为了阐明E-钙粘蛋白在三维球形体培养维持MSCs干性中的作用机制，本项目拟：①明确E-钙粘蛋白对三维球形体培养MSCs干性维持的作用；②评价E-钙粘蛋白复合体的胞内连环蛋白对三维球形体内MSCs干性的影响；③研究连环蛋白影响三维球形体培养MSCs干性维持的作用机制；④探讨连环蛋白调控基因对E-钙粘蛋白的反馈调控作用。.本研究以微重力生物反应器内球形体培养的ADSCs为研究对象，取得的重要实验结果、发现及科学意义如下：.①抗体中和或shRNA下调E-钙粘蛋白，可抑制反应器内ADSCs球形体的形成，降低细胞的增殖能力、克隆形成率、干性相关基因表达和多向分化潜能等干性指标；上调E-钙粘蛋白表达，可促进反应器内ADSCs球形体的形成，提高ADSCs干性；表明E-钙粘蛋白对球形体培养MSCs干性维持发挥关键作用。.②抑制E-钙粘蛋白复合体形成显著降低球形体培养ADSCs的干性，提示E-钙粘蛋白复合体的形成有利于球形体培养ADSCs干性的维持；上调E-钙粘蛋白表达可显著提高球形体培养ADSCs细胞膜上β-连环蛋白的表达；上调β-连环蛋白表达可显著提高球形体培养ADSCs的干性，下调β-连环蛋白表达可造成球形体培养ADSCs的干性降低。.③利用ChIP技术和双荧光素酶报告基因系统发现β-连环蛋白通过与OCT4启动子结合调控球形体培养ADSCs的干性维持。随着E-钙粘蛋白表达的增加，可反馈调节E-钙粘蛋白的基因Snail和Slug逐渐升高，提示Snail和Slug可能反馈调控E-钙粘蛋白表达的平衡。.④阐明了MSCs通过重塑巨噬细胞表型诱导血管内细胞功能恢复，以提高糖尿病皮肤创面修复速度的作用机制。.因此，本研究从三维立体的新视角阐述MSCs干性维持的作用机制，为探索既能维持干细胞干性又适合细胞大量扩增的体外培养方法提供实验依据，为MSCs的临床转化奠定基础。