Pea3/Scx调控间充质干细胞促进骨-肌腱结合部愈合的机制研究

The scar healing is the main way for repair after bone tendon junction (BTJ) injury. Therefore, it is difficult to restore the original fiber cartilage structure and the rate for injury again is relatively high. As one of well recognized tendon specific markers, scleraxis (Scx) has a critical role on differentiation and development of tendon. However, the mechanism on regulation of Scx expression remains poorly unknown. Our previous study demonstrated that both Pea3 and Scx were up-regulated in the model of rotator cuff injury repair. Moreover, scx expression was attenuated after interfering with Pea3 expression. Accordingly, We hypothesize that Pea3 can promote the differentiation and development of MSCs through regulating Scx, thereby benefiting for the repair of rotator cuff injury. This study aims to explore the effect and potential mechanisms of Pea3 in promoting MSCs differentiation and development by modulating Scx. The study mainly include the following parts: 1) detailed mechanism for Pea3 promoting differentiation and development of MSC mediated by Scx in the cellular level; 2) confirmation of the effects for Pea3 on the differentiation and development of BTJ by in vivo experiments; 3) discussion of feasibility and application of Pea3 on BTJ regeneration in a rat model of shoulder cuff injury. Thus, our study will further uncover the mechanism for the expression of Pea3/Scx on the regeneration of BTJ , finally resulting in substantial survival benefit.

骨-肌腱结合部（BTJ）损伤修复后以瘢痕连接愈合，难以恢复原本的纤维软骨性结构，再损伤率高。转录因子Scx对肌腱的发育、分化有着至关重要的作用，然而Scx表达的调控机制尚不清楚。我们前期研究显示：在肩袖损伤修复模型中多瘤病毒增强子激活剂3（Pea3）和Scx的表达具有协同性，干扰Pea3的表达后Scx的表达也明显降低。因此我们推测，Pea3可通过调控Scx促进间充质干细胞（MSCs）分化发育，从而促进腱骨愈合。本项目拟深入探索Pea3通过Scx介导促进MSCs分化的作用及机制：细胞水平研究Pea3对Scx表达的作用及对MSCs分化发育的调控；体内实验验证Pea3对BTJ分化发育的影响；并采用肩袖损伤修复动物模型探讨Pea3调控MSCs分化，促进BTJ再生的可行性。本研究将揭示Pea3/Scx的表达在BTJ损伤愈合过程中的作用和机制，为调控干细胞分化而促进BTJ的愈合提供理论依据和潜在方案。

一、项目背景：肩袖损伤是运动医学中的常见病，肩袖损伤修复后只能以瘢痕连接愈合，难以恢复原本的纤维软骨性骨肌腱结合部（BTJ），有损其机械强度和最大载荷，再撕裂率高。目前研究发现合理调控间充质干细胞增殖和向肌腱分化可以起到促进肩袖腱骨愈合，提高术后功能、降低再撕裂率的作用。.二、主要内容及结果：（1）我们通过CCK-8,显微镜细胞形态观察发现Scx可以抑制BMSCs增殖能力，上调腱系相关基因（Col1a1、Tnmd、Dcn）的表达。（2）Scx-BMSCs三系分化染色显示其软向骨细胞分化能力显著下降、向脂肪细胞分化能力显著下降、向成骨细胞分化能力无显著差异，同时软骨形成关键基因Sox9表达明显受抑制，骨系分化基因Runx2和脂肪系分化基因Taz表达变化无明显统计学差异。（3）RNA高通量测序结果提示Scx通过促进了BMSC中Murc的表达，抑制了CD 36、Igf 1的表达。（4）Pea3的过表达可以提高C3H10T1/2细胞增殖能力，Pea3的激活可以上调腱系相关基因Col I、Col III及Scx的表达，然而对Tnmd蛋白的表达无明显调控作用.（5）FGF2通过上调Pea3来促进Scx的表达。.三、结论及意义:本研究发现，Scx可以抑制间充质干细胞增殖及向软骨、脂肪方向分化，上调肌腱分化相关因子,促进其向肌腱分化，是调控间充质干细胞向肌腱分化、促进腱骨愈合的重要因子。同时，Pea3的激活可以促进间充质干细胞的增殖与上调肌腱分化相关因子，其中Pea3促进Scx的表达上调可能是其调控间充质干细胞增殖及向肌腱分化的原因。本研究为揭示间充质干细胞促进腱骨愈合的分子机制提高了有力证据，为干细胞治疗促进腱骨愈合提供了理论基础。