量化受控的胞间接触调控骨髓间充质干细胞分化的规律及作用机理研究

Cell-cell contact is one of the important extracellular factors that affect differentiation of the bone marrow mesenchymal stem cell (BMSCs), but the specific characteristics and mechanism of regulation are still not clear enough. This study will construct a suitable micro-pattern for long time cell adhesion and achieve the precise control of intercellular contact. This study is supposed to be done in the following three aspects: Firstly, we are going to study the relationship between the extent of intercellular contact or contact symmetry and the level of one-way induced differentiation or the directions of two-way co-induced differentiation. This is to verify the hypothesis that extent of intercellular contact can promote its differentiation capacity and symmetry of intercellular contact can influence the direction of differentiation. Secondly, a digital image processing program is needed to develop to extract geometric information of cytoskeleton from confocal microscopic images. The difference of cytoskeletal structures induced by different extent of intercellular contact is analyzed to test the assumption that intercellular contact can strengthen intracellular cytoskeleton. Lastly, experiments are designed to verify the guess that gap junctions can directly affect stem cell differentiation without relying on cytoskeleton.In these experiments, the major protein of anchoring junction and the contractility of cytoskeleton are blocked simultaneously. Other experiments, in which gap junction and cytoskeleton are blocked, are designed to verify the hypothesis that anchoring junction must depend on cytoskeleton to affect differentiation. This project will help to clarify the rules and mechanisms of intercellular contacts affecting the differentiation of stem cells and advance the process of clinical applications of BMSCs, and has important basic scientific significance.

细胞间接触是影响骨髓间充质干细胞分化的重要胞外因素，但具体的调控规律和作用机制仍有待研究。本研究建构了适合细胞长时间粘附的微表面图案，实现了对胞间接触的精确控制。此项目拟展开三方面研究：1)研究胞间接触程度、接触对称性对干细胞单向诱导分化程度及双向共诱导分化方向的影响规律。验证胞间接触程度越高越能促进其各向分化能力及胞间接触对称性能影响其分化方向的假设。2)编写数字图像处理程序，实现对共聚焦显微图像中细胞骨架几何信息的提取。分析不同胞间接触程度下细胞骨架结构差异，验证胞间接触能加强细胞骨架结构的猜测。3）设计同时阻断锚定连接和细胞骨架的实验，验证间隙连接可以不依赖细胞骨架而直接影响干细胞分化的猜想，设计阻断间隙连接和细胞骨架的实验，验证锚定连接必须依赖细胞骨架进而影响其分化的假设。此项目将有助于阐明胞间接触影响干细胞分化的规律和机制并推进BMSCs的临床应用进程，具有重要的基础科学意义。

细胞间接触是影响骨髓间充质干细胞分化的重要胞外因素，但具体的调控规律和作用机制仍有待研究。细胞的几何特征是内在生理特性表征，但一定程度上也能影响其生理功能；细胞几何特征必然被动的受其力学状态的影响，但也能主动地去适应其力学状态。本项目从粘附细胞的几何特征与其生理与力学状态之间的关系入手，展开三方面研究：分别为：第一，实现了在微模式化基底上培养细胞的技术，完成了用TUNEL法和磷脂丝氨酸外翻分析法分别检测细胞凋亡的实验，发现粘附面积增加有利用降低细胞凋亡，粘附形状圆度降低也有利于降低细胞凋亡。另外，实验中还发现了小岛内粘附细胞的泊松分布特点和小岛内双细胞极化粘附现象，具体细节和机理有待进一步研究。第二，本文建立的IFS算法可用于定量分析亚细胞尺度的曲线纤维网络结构，识别纤维间的连接关系，提取单根纤维的长度、方向、位置等拓扑信息，呈现整个网络中所有纤维不同拓扑性质的分布特征。与同类算法相比，IFS算法的最大优点在于它在完成了对曲线网络中纤维结构的识别和分割后，还能够不依赖人为的干预和判断完成对显微图像中曲线网络结构的定量拓扑分析。面对分析如细胞骨架等曲线纤维网络结构的生物显微图像时，我们往往会基于目测去得出一些定性的结论，而定量的分析能更有效地帮助我们找到更精确的量化规律，甚至不易直接察觉的内在关系，因此能很大程度地提高我们获取大量生物实验图片背后隐藏着的知识和规律的能力。第三，本文通过准三维显微观测系统，获得了部分粘附细胞在剪切流作用下构型变化的实验图像，并由此建立了反映细胞剪切流相互作用的三维流固耦合有限元模型。在率定出细胞粘弹性系数的基础上，从数值模型中获得了剪切流作用下部分粘附细胞的受力和变形特征。

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