肝细胞生长因子对肌腱干细胞生物学特性的调控作用及其促进肌腱优质愈合的分子机制

The healing of tendon is slow after injury. The biological characteristics of healing tissue with scar are inferior to normal tendon tissue, which lead to impairment of function. Hepatocyte growth factor (HGF) is a growth factor which appears in injury site, can promote cell proliferation, differentiation, migration and matrix remodeling for anti-fibrosis. Our preliminary study results show that: HGF can accelerate the tendon healing speed, and can resist tendon fibrosis by the transforming growth factor beta1 (TGF- beta1) induced. In the present research, we hypotheses that HGF could optimize the repair of injured tendon by regulating tendon stem cells. Moreover, HGF may play important role in the restoration of structure and function after tendon injury by modulating myofibroblast transdifferentiation and the balance of MMP/TIMP system during tendon healing process, which result in optimized regeneration. We hypotheses that HGF promote tendon healing by regulating above mechanisms and interfering with the paracrine of tendon stem cells, and adjust the niches of tendon stem cells, which served as the breakthrough point in this study. We will further explore the molecular mechanism and c-Met, MAPK signal pathway of HGF improving the histological, biochemical and biomechanical properties of the healing tendon. Tendon stem cells and tenocytes cultured in vitro and the animal model of patellar tendon injury were used in the experiment. The result of this research may provide a new treatment strategy and theoretical basis for improving the quality of the healing tendon tissue.

肌腱损伤后常呈瘢痕愈合，而且愈合速度慢，丧失正常的生物学特性，严重影响功能。肝细胞生长因子（HGF）具有多种生物学功能。我们前期研究表明：HGF能抵抗由于TGF-β1诱导的肌腱肌成纤维细胞（MFB）过度增生和细胞外基质过度合成，能改善韧带和肌腱的愈合质量；初步还研究了肌腱干细胞(TSC)具有的腱性分化潜能。本课题设想HGF可以通过调控TSC生物学特性来优化愈合微环境和愈合过程，改善TSC促进损伤肌腱的修复活动；对抑制MFB转分化以及MMP/TIMP系统平衡表达可能具有调节作用，进而达到优质愈合。本课题以HGF是否通过调控TSC的旁分泌并进而调节TSC生长的微环境为切入点，拟利用大鼠肌腱干细胞、肌腱细胞体外培养及膑腱急性损伤动物模型，探索肌腱优质愈合的内在分子机制，同时对可能涉及的c-Met, MAPK等信号通路进行探索。本研究的成功实施将为提高肌腱愈合质量提供新的治疗策略和理论依据。

肌腱和韧带损伤是常见的运动损伤，肌腱愈合的潜力有限以及目前外科治疗手段不完善，促使我们研究可替代的治疗方式以促进肌腱组织再生。我们前期研究发现：肝细胞生长因子（HGF）能够抑制TGF-β1诱导的肌成纤维细胞(MFB)过度增殖、MFB过度转分化以及ECM过度合成作用，从而对抗TGF-β1过度产生导致的纤维化和瘢痕愈合。.本研究通过细胞学实验、动物实验，借助于分子生物学技术、病理学技术等手段，着重研究HGF对肌腱干细胞（TSC）的调控以及这种调控对肌腱细胞生物学行为的影响，同时通过HGF转染，研究TSC生长微环境的改变对肌腱愈合的影响，并对可能涉及的信号通路进行研究；目的是改善肌腱愈合质量。.本课题获得的成果为：（1）损伤肌腱组织中HGF表达增加，但是持续时间有限；（2）外源性HGF能够促进TSC增殖、迁移，抑制TGF-β1介导的TSC过度成骨分化，从而减轻异位骨化造成的肌腱不良愈合；（3）HGF通过调节TSC生长的微环境，产生的条件培养液能优化愈合期MFB的生长，从而为促进肌腱优质愈合提供另一种手段。HGF通过转染TSC能明显提高肌腱损伤动物模型的运动能力；（4）MAPK/ERK、PI3K/Akt和BMP信号通路参与了这些过程。（5）此外，我们发现不论是脂肪源性干细胞、骨髓间充质来源干细胞以及TSC，它们在促进大鼠肌腱愈合过程中，都不能避免异常骨化地发生，而且干细胞在肌腱损伤修复过程中的成骨细胞分化对于早期改善肌腱组织张力、刚性，改善鼠运动能力还有帮助。其中腱性分化则以TSC最强。.本课题的科学意义在于：HGF可以通过调控TSC生长、分化、以及微环境改变，来改善损伤肌腱和韧带愈合的质量。为提高肌腱愈合质量提供新的治疗策略，因而具有重要的研究价值，其广阔的应用前景让我们充满期待。也为今后开发研制促进肌腱优质愈合药物及构建组织工程肌腱奠定理论基础和研究平台。