P311在烧伤创面表皮干细胞向肌成纤维细胞转分化中的作用与机制研究

Scar formation is still a great challenge not only for patients but also medics. The mechanism of scar formation is not illustrated clearly yet, therefore either the effect of prevention or treatment is very limited. Myofibrolast is the main functional cell in scar formation, which is believed to be derived from fibroblast. In our last project and primary experiment, it was found that epidermal stem cell maybe was another important source of myofibroblast in scar formation after burn injury. And our primary research also show P311, a new found gene, probalblely played a vital role in the transdifferentiation of epidermal stem cell to myofibroblast. Moreover, from our previous study and literatures,it is suggested that P311 facilitate epidermal stem cell to transdifferentiate to myofibroblast through TGFβ/Smad-Rho GTPase/Rho kinase signal pathway. Therefore, we think there may be an new hypothesis of "burn-P311- - -TGFβ/Smad-RhoGTPase/Rho kinase　-　transdifferentiation of epidermal stem cell to myofibroblast-scar formation". In this project, we expect to prove our hypothesis by gene knockout animal, gene transferring, viable cell station, pulldown and other techniques. With the accomplishment of this project, it will be stated that epidermal stem cell should be another important source of myofibrobalst by transdifferentiation and P311 plays a vital role in this process, which will rich and consummate the mechanism of scar formation and supply new target and clue for the prevention and treatment of scar after burn injury.

讫今，皮肤瘢痕形成机理仍不十分清楚，致其预防与治疗效果均十分有限。肌成纤维细胞是瘢痕形成中最主要功能细胞，一般认为它来源于成纤维细胞。我们在上一基金及前期研究中发现，表皮干细胞通过转分化可能是创面中肌成纤维细胞的另一重要来源，并发现，P311可能在其中起着重要作用。文献与我们前期研究结果进一步提示，P311可能是通过TGFβ/Smad-Rho GTP酶/Rho激酶信号途径促进表皮干细胞向肌成纤维细胞转分化。为此，我们提出 "烧伤- P311-TGFβ/Smad-Rho GTP酶/Rho激酶-表皮干细胞向肌成纤维细胞转分化-瘢痕形成" 的新假说。拟通过转基因技术、基因敲除动物、活细胞工作站等技术进行体内外实验研究证实我们的设想。本课题的顺利完成，将明确表皮干细胞的转分化是烧伤瘢痕形成中肌成纤维细胞的另一重要来源，这将进一步丰富与完善瘢痕形成机理，并为皮肤瘢痕的预防与治疗提供新靶点与新线索。

烧伤、创伤、手术、糖尿病等引起的皮肤创面是常见的临床问题之一，创面愈合后不可避免会产生不同程度的瘢痕。表皮干细胞和肌成纤维细胞在创面愈合中发挥关键作用，一般认为肌成纤维细胞还参与了瘢痕形成，并认为肌成纤维细胞主要来源于成纤维细胞，而较少有研究发现表皮干细胞可通过转分化而形成肌成纤维，进而参与瘢痕形成。本研究旨在明确P311对表皮干细胞向肌成纤维细胞转分化的影响及其机制。本研究首次证实，P311是一种新的表皮干细胞向肌成纤维样细胞转分化的调控因子。本研究发现，转分化后的细胞I型胶原和MMP2蛋白合成增加、迁移能力增强，但并不具有收缩功能。因此，P311可以促进表皮干细胞转分化为肌成纤维样细胞，而不是肌成纤维细胞。P311可以激活TGFβ1/Smad通路，TβRI/II抑制剂LY2109761和Smad3 siRNA均可阻断P311引起的转分化，而外源性TGFβ1可以恢复P311敲除小鼠表皮干细胞的转分化能力。从而证实，P311可通过TGFβ1/Smad通路促进表皮干细胞向肌成纤维样细胞转分化。进一步研究发现，P311通过增强TGFβ1启动子的甲基化抑制TGFβ1转录，通过结合并激活TGFβ1 5’-UTR和3’-UTR促进TGFβ1的翻译。烧伤创面局部的炎性因子（如IL1β、TNFα、IL6等）和缺氧微环境可能是引起表皮干细胞P311表达升高的主要因素。敲除P311可导致烧伤创面愈合延迟。综上，本研究结果不仅可以明确表皮干细胞是皮肤创面愈合中肌成纤维细胞的另一重要来源，更为重要的是，发现了一种新的皮肤创面愈合机制，即：烧伤-炎性因子和缺氧-P311 -TGFβ1-Smad2/3-表皮干细胞向肌成纤维样细胞转分化-MMP2和I型胶原分泌增多-迁移能力增强-创面愈合加快。研究结果丰富和完善了P311的生物学功能和烧伤创面愈合机理，同时为创面愈合的基础研究和临床治疗提供新靶点和新线索。