真菌来源透明质酸对人羊膜间充质细胞的增殖及其向软骨、成骨细胞分化的影响与作用机制

Considerable progress has already been made in clinical applications of stem cell research, but still need discovering the regulatory networks of proliferation & differentiation (P&D) in the stem cell development process, a key scientific question for clinical benefit. Human amniotic mesenchyal cells (hAMCs) are recognized as new source for clinical application of stem cells. Hyaluronan (HA) is a good regulator of stem cell P&D. Also, HA, a major component of extracelluar matrix, has natural predominance to regulate the stem cell P&D due to immunogenicity. However, little is known about the influences of HA on the P&D of hAMCs and their acting mechanism. Based on our results and the related research progress on stem cell, we presume that HA can modulate hAMCs proliferation and to differentiated into different cell types such as chondroblast, and osteoblast, in which Wnt signalling plays a vital role in the developmental process. To verify the hypothesis, a series of experimental techniques, including immunohistochemistry, immunoblotting, RT-PCR, RNAi, and Genechip, will be employed to investigate the effects of HA on the P&D of hAMCs and the corresponding changes properties of gene and protein in Wnt pathway, and elucidate their acting mechanisms for the first time. The findings will replenish and enrich the intracellular regulatory networks associating with the P&D of hAMCs, give the further evidences that hAMCs are served as the best seed cells for bone and cartilage tissue engineering. Also, facilitate to repair defects in bone and cartilage, and to construct the implant for bone and cartilage tissue engineering by the combination of hAMCs and HA. At the same time, the findings also provide a new pathway for the exploitation of the fungal-derived HA.

人羊膜间充质细胞(hAMCs)是最具开发潜力的干细胞新资源。透明质酸(HA)有良好的调节增殖分化能力。作为胞外基质(ECM)的主成分，HA用作干细胞增殖分化的调控因子有先天优势。但HA对hAMCs增殖分化的影响及作用机制尚不明确。基于我们的前期研究及文献报道，推测HA是hAMCs增殖及向软骨、成骨细胞分化的潜在调节剂，且Wnt信号在其调控网络体系中扮演了重要角色。为证实这一假说，采用免疫组化、免疫印迹、RT-PCR、RNA干扰、基因芯片等技术，在细胞、基因及蛋白水平上，探讨真菌来源HA对hAMCs的增殖以及分化成软骨、成骨细胞的影响，并揭示Wnt信号通路基因蛋白的变化规律，阐明其作用机制，为完善hAMCs体外增殖分化调控网络体系，及其作为骨及软骨组织工程最佳种子细胞提供实验依据；为hAMCs联合HA用于骨病损细胞治疗、骨及软骨组织工程种植体的构建奠定基础；给真菌来源HA的开发提供了新途径。

干细胞研究及其转化应用为许多重大疾病的有效治疗提供了新的思路和工具。但是，其应用于临床治疗，最大的挑战在于稳定的细胞来源、足够的移植细胞量，以及细胞定向分化技术，直接影响到干细胞及相关产品的安全性、有效性与可控性。骨病损是严重危害人类健康的常见病、多发病，干细胞用于骨病损的修复与功能重建有巨大潜力。本项目以理想的干细胞新资源——人羊膜间充质干细胞(hAMSCs)为研究对象，重点针对其临床应用亟待解决的增殖分化科学问题，选取胞外基质主成分透明质酸（HA）为干预因子，研究其对hAMSCs增殖及向成骨、软骨细胞分化的影响及作用机制，以及治疗骨性关节炎软骨缺损疾病模型的潜力。发现了300kDa透明质酸在0.6mg/mL时能显著促进hAMSCs的增殖，且不影响其细胞表面标志及分化潜能等生物学特性，其促hAMSCs的增殖的作用机制主要涉及经典Wnt/β-catenin信号通路；发现HA与常规成骨诱导剂联合作用于hAMSCs，可显著协同增强hAMSCs向成骨细胞分化的效率，其协同促成骨分化的作用机制涉及TGF-β信号通路；HA具有促进hAMSCs向软骨细胞分化的作用，其与常规成软骨诱导剂联用，有明显的协同增强效应，DLX5、HFE2、RASL11B和ICAM4等基因在其中扮演了重要角色; 而且，HA与hAMSCs联用移植治疗骨性关节炎软骨损伤模型，有显著的协同增强关节软骨损伤修复的作用，这与促进hAMSCs植活、分化，增强软骨相关因子、抗炎因子和骨保护素的表达水平，并抑制炎性因子表达有关。这些研究结果为完善hAMSCs体外增殖分化调控网络体系，及其作为骨及软骨组织工程最佳种子细胞提供实验依据；为hAMSCs联合HA用于骨病损细胞治疗、骨及软骨组织工程种植体的构建奠定基础，并给HA的开发利用提供了新途径。