间充质干细胞和巨噬细胞在仿生微环境中的双向调控机制研究

In vivo, the crosstalk between mesenchymal stem cells (MSCs) and macrophage (Mø) plays an important role in tissue homeostasis, and in the occurrence and development of diseases. Their crosstalk is tightly modulated by the microenvironment cues, thus understanding their bidirectional regulation mechanisms in a specific microenvironment is critical for the treatment of immune disease and tissue injury. In this study, a biomimetic microenvironment is constructed using human platelet lysate (HPL) and surface with different micro-nanotopographical cues, as previous study found that HPL can be successfully used for MSCs expansion as a substitute for FBS and that specific surface micro-nanotopography can boost the differentiation of MSCs towards specific cell type. The impact of different microenvironment on MSCs and Mø behaviors, functions as well as their bidirectional regulatory interactions are investigated. Firstly, the cytokine profiles of MSCs obtained with different microenvironment are exploited via a high-throughput cytokine array kit. The polarization effect of cytokines secreted by MSCs on Mø (M1/M2) is investigated to demonstrate the plasticity of MSCs in immunomodulation. Understanding the intricate secretory activities of MSCs in different microenvironment is critical to harness their clinical potential. Secondly, the impact of biochemical and biophysical cues in the biomimetic microenvironment on the polarization and on inflammatory cytokines (IL-10, TNF-α) secretion of Mø is investigated. Further, the effect of inflammatory factors secreted by Mø on MSCs differentiation and gene expressions is investigated to explore the role of Mø on tissue repair and reconstruction. The aim of this study is to develop a biomimetic microenvironment that can maintain specific cellular function of MSCs for clinical applications and find a biochemical and biophysical property that can enhance specific bioactivity of implant materials, and finally provide therapeutic implications for immune diseases.

间充质干细胞(MSCs)与巨噬细胞(Mø)间的双向调控作用对人体组织动态平衡的维持与疾病的发生和发展都具有重要意义。本项目采用人源可溶性因子结合微纳米结构表界面在体外构建仿生微环境，并展开MSCs和Mø的双向调控研究：一，通过高通量方法检测不同微环境中MSCs分泌因子的成分及该成分对Mø表型转化(M1/M2)的作用，揭示仿生微环境中的生化和物理信号对MSCs免疫调控特征的可塑性，为提高MSCs对免疫性疾病的治疗效果提供优化方案。二，探索微环境中的生化和物理信号对Mø表型及炎性因子(IL-10,TNF-α)分泌的影响，并通过炎性因子对MSCs分化和基因表达的影响，阐明不同表型的Mø在组织修复与重建过程中的作用机制，从而为设计移植材料表面物理化学组成提供实验依据。本项目通过新型的体外仿生微环境探索MSCs与Mø相互调控作用，揭示体内炎症反应与组织修复的关系，为炎性疾病的治疗提供了解决方案。

近年来，干细胞领域的研究进展为退行性疾病的治疗带来了希望。在多种干细胞类型中，间充质干细胞(MSC)因具有强大的组织修复、再生功能以及免疫抑制特性，而成为当前干细胞研究和应用的首选细胞类型。然而，体外培养的干细胞通常会由于缺乏有效模拟其微环境的平台而失去干细胞性能。干细胞所在的特定微环境包括细胞和非细胞成分，这些成分的物理结构特征和生物化学成分等共同维持干细胞性能并调控其分化方向。常规应用于MSC扩增的培养基是添加胎牛血清（FBS）的基础培养基，FBS中含有丰富的生长因子、激素、黏附蛋白和其他生物活性因子能有效维持MSC的自我更新能力和干性功能。然而，由于FBS来源于动物包含异源成分并具有传播病原体的风险而限制了其在人类疾病中的临床治疗。人血小板裂解液（hPL）因含有丰富的生物活性因子能更好地提高MSC的自我更新和分化潜能而被认为是FBS的有效替代品。然而，hPL对不同组织来源MSC的调控机制和效率尚不明确。因此，本研究系统性研究了hPL和FBS作为培养基添加成分对MSC黏附、增殖和分化的影响，并利用转录组测序和分子生物学手段对其中的调控机制做出探索，结果发现hPL相对于FBS更有效地促进MSC的增殖和分化。除生物化学信号外，微环境中的物理特性如力学性能和拓扑结构特征等都对细胞行为有着不可替代的作用。然而，常规二维塑料培养皿和单一组成的合成材料等并不能模拟干细胞原始微环境的复杂性和多样性。鉴于此，本研究是基于hPL取代FBS作为细胞体外扩增培养基添加成分的潜能，并结合当前制备多种纳米拓扑结构的工程技术手段，在体外构建一个无异种添加成分的仿生微环境。以此微环境为基础，探索体外的不同培养条件对MSC自我更新、分化命运以及旁分泌功能的调控机制。本研究的最终目标是通过改变体外培养微环境实现对细胞特性和分泌蛋白组成分的优化，最终为细胞治理方案的开发、合理应用和移植材料表面结构的设计提供实验和理论依据。

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