局部高密度培养扩增骨髓血管内皮祖细胞及其机理

Expansion of adult stem cells and maintenance of their "stemness" in culture is still a big challenge in this field. It has been well known that homeostasis and self-renew of stem cells depend on the stem cell "niche" in the body. The communication between stem cells and their surrounding cells plays an important role in the niche. Conventional cell culture methods disrupt the cell-cell communication, results in the failure of stem cell expansion in culture. We proposed that local high density cell culture, which maintains cell-cell interaction, could mimic the stem cell niche and therefore expand stem cells in culture. For expansion of bone marrow endothelial progenitor cells (EPC), our previous study showed that the high density culture system could efficiently expand EPCs. The purpose of this study is to investigate the in vivo blood vessel formation activity of these cells and to evaluate their therapeutic potential.In addition, the basic mechanisms of how high density culture could maintain EPC in vivo will be analyzed. Finally, this work will develop a novel system for EPC expansion and establish an in vivo model for studying stem cell niche.

干细胞体外扩增和干性维持是本领域的研究热点和难点，已知体内干细胞特性维持与自我更新依靠其所处的微环境，其中干细胞与周围细胞的接触与信息交换起着重要的作用，而传统体外干细胞培养方法破坏了细胞间的接触与信息交换，降低了扩增效率。由此我们提出了局部高密度细胞接种的设想，以体外扩增骨髓血管内皮祖细胞为例，通过高密度接种骨髓细胞的方法来维持细胞间的接触，以此模拟骨髓微环境，达到扩增骨髓血管内皮祖细胞的目的。前期研究结果提示局部高密度培养能显著提高血管内皮祖细胞的扩增效率并维持其干细胞特性，本项目将进一步通过体内外实验证实扩增细胞的成血管能力与治疗应用价值，并通过基因芯片分析等手段，发现并验证与高密度培养相关的特异性分子，阐明高密度培养维持血管内皮祖细胞特性的可能机制。该研究将解决骨髓血管内皮祖细胞体外扩增难题，并为研究干细胞微环境建立重要的体外研究模型。

干细胞体外扩增和干性维持是本领域的研究热点和难点，而维持干细胞与周围细胞的接触与信息交换起着重要的作用。我们提出了局部高密度细胞接种的设想，建立了以高密度接种骨髓细胞维持细胞间接触从而达到扩增骨髓血管内皮祖细胞的新方法。局部高密度培养扩增后的骨髓细胞经细胞表型及功能分析，证实其富含血管内皮祖细胞；进一步通过动物体内下肢缺血治疗模型，证实扩增的细胞具有促血管新生能力；通过动物肝硬化治疗模型，证实其具有促进肝脏修复和抗肝纤维化的作用，显示了高密度扩增细胞的临床应用前景；通过基因芯片分析，发现并验证了与高密度培养相关的生长因子表达变化，部分阐明了高密度培养维持血管内皮祖细胞特性的机制。该研究建立了骨髓血管内皮祖细胞体外扩增新技术，并为体外研究干细胞微环境建立了新的研究模型。