Src介导的细胞有丝分裂方向调控在皮肤创伤修复过程中的作用

Oriented cell division, which is determined by the axis of a mitotic spindle, plays an essential role in controlling cell fate choice, tissue architecture and tissue morphogenesis. The mechanisms that determine the axis of division in the context of a tissue are poorly understood. Recent studies evidenced that the proper control of cell division axis rely on integrating intrinsic polarity cues, and extrinsic, such as cues associated with the cell's contacts to its adhesive microenvironment. Cell shape is another determinant for the orientation of cell division machinery. In previous study, we used micro-contact printing and EFs (electric fields) to control the spatial distribution of the ECM (extracellular matrix) on the substrate and demonstrated that Src is an important regulator in decoupling the interaction of cell geometry from cell division orientation. Src manipulate the accumulation and local density of adhesive proteins to the sites of cell-matrix attachment to affect the force balance on the spindle pole and subsequently induce the spindle misorientation. As the first oncogene to be discovered, Src has a critical role in cell adhesion, invasion, proliferation, survival, and angiogenesis during tumor development. Cancer development and wound healing share common features, implying that the Src-induced cell division orientation may also play roles in wound repair and regeneration. In this study, we first use the expression of Src as a reference to evaluate the distribution of cell division in wounds with different depth and different healing phase, aiming to understand how cell division happens during wound healing and its relationship with Src activation. We then use high-resolution multiphoton microscopy combined with cell lineage tracing to visualize the migration, cell division orientation, differentiation, and cell-fate determination of follicle stem cells during skin wound healing and tissue regeneration in three dimensions, which might shed new insight on the roles of Src kinase activity in regulating wound repair and tissue regeneration. Our study also provides a novel target for therapeutic interventions during skin wound healing to rebulid the histological architecture and kinetics in reconstructed tissues, to restore normal function to damaged tissues, and ultimately reach the goal of perfect skin regeneration.

细胞有丝分裂的方向不仅关系到细胞增殖过程中子代细胞的排列方式，同时对细胞分化、组织器官结构形成具有决定性的作用。在前期的工作中我们采用表面微图形印迹技术和施加外源性电场来界定细胞的几何形状，发现细胞的外在几何形状仅仅是影响细胞有丝分裂方向的因素之一。原癌基因Src在调控细胞有丝分裂方向的过程中具有重要作用，本研究在前期工作的基础上，首先确定Src的表达对重建组织内细胞排列方向和功能取向的影响。随后，采用激光共聚焦断层扫描合并荧光细胞示踪技术从三维角度分析毛囊干细胞在创伤修复过程中的迁徙途径、分裂方向及命运转归。本研究抓住了创伤修复过程中的一个关键性科学问题，研究结果在理论和技术上的突破为精确调控修复过程中再生组织的时序性，改善其力学状态指出了一条新的解决途径。同时，本研究也为促进皮肤组织修复与重建过程中细胞功能的正常转归，实现创伤后皮肤组织的无延迟、无瘢痕修复开辟了一个新的视角。

细胞有丝分裂的方向不仅关系到细胞增殖过程中子代细胞的排列方式，同时对细胞分化、组织器官结构形成具有决定性的作用。研究表明，细胞外基质的空间分布不但可以约束细胞的外在几何形状，其作用还可以超越细胞的几何形状，指导细胞的有丝分裂方向。特别是我们的前期工作表明，原癌基因Src在调控细胞与ECM的黏附过程中具有重要作用。本课题在前期工作基础上进一步研究发现：（1）Src底物蛋白Par3在正常、创缘及瘢痕组织中呈现规律性极性分布。尤其是Par3在基底层细胞中的极性表达不仅与该皮肤标本的组织学结构有关，同时还决定了表皮基底层细胞的有丝分裂方向；（2）通过HE、Masson病理染色结合免疫荧光检测，从多个水平证明细胞外基质的结构异常能够影响表皮基底层细胞与基底膜的黏附作用，进而影响细胞极性蛋白Par3在表皮基底层细胞内极性分布。细胞外基质的结构异常不仅是基底层细胞有丝分裂方向紊乱的始作俑者，同时还从体内、体外两个水平决定了表皮细胞的分化谱系，最终导致终末分化标记CK10在表皮细胞内的表达上调；（3）过检测α-SMA 的表达进一步发现位于真皮乳头层内的成纤维细胞是影响基底膜形成，调控表皮基底层细胞与细胞外基质黏附作用的关键因素之一。这些细胞的培养上清液不但可以诱导Par3在表皮细胞中的表达，还能够上调黏附相关分子Integrin-β4、Cortactin等在表皮细胞中的表达水平，增强表皮细胞与外界环境的黏附作用，影响其有丝分裂的方向；（4）开展新探索以β2-AR信号通路为介入点，研究β2-AR及其下游信号分子诱导角质细胞表型变化及命运转归的相关分子机制，阐述神经-内分泌因素影响皮肤创伤修复与组织再生的调控网络。本课题抓住了当前学科领域的关键问题，研究结果不仅为突破皮肤创伤修复研究领域的瓶颈，建立个体化的治疗方案打下基础，同时也将为多层次、多靶点地激活创面内各细胞组分的潜能，促进损伤后多种组织的原位同步再生提供理论参考。