低表达CSMD1促进底物磷酸化在增生性瘢痕中的作用及其机制研究

The mechanism of scar formation is complex, and its molecular biological mechanism is unknown, which severely limits the development of targeting therapy. The latest genome-wide association study (GWAS) showed that the hypertrophic scar was significantly related to three single nucleotide polymorphisms (SNP) in CSMD1 gene; Our group's research in the mRNA and protein levels preliminarily found that CSMD1 was significantly decreased in hypertrophic scar; The possible regulation of downstream Smad/ TGF-beta pathway is also an important pathway of scar formation; All above suggests that CSMD1 is a key gene in the occurrence and development of scar. CSMD1 is a transmembrane phosphorylation protein which is the most common target protein for the targeting treatment . On this basis, to promote “the low expression of CSMD1 in hypertrophic scar as the hypothesis”, by means of tissue microarray, lentivirus cell model, protein liquid chromatography mass spectrometry technique and protein chip method, our group will explore specific substrates and signal transduction pathway of CSMD1 in hypertrophic scar from tissue, cell, protein and gene levels without deviation, in order to find the target of the hypertrophic scar targeting therapy, and finally explore a new method for the treatment of scar.

瘢痕形成的分子生物学机制不明，严重限制了靶向治疗的开发。最新全基因组关联分析（GWAS）表明三个同位于CSMD1基因的单核苷酸多态性（SNP）与增生性瘢痕显著相关；项目组预初研究发现增生性瘢痕中CSMD1在mRNA及蛋白水平均显著下降；其可能调控下游Smad/TGF-β通路亦是瘢痕形成重要信号通路；以上提示CSMD1是瘢痕发生发展的关键驱动基因。而CSMD1基因所表达的跨膜磷酸化蛋白是最为常见靶向治疗的靶点蛋白。在此基础上，本项目将以“CSMD1低表达促进增生性瘢痕发生”为假说，借助组织芯片、慢病毒干扰细胞模型、蛋白质液相质谱技术以及蛋白芯片等手段，从组织、细胞、蛋白、基因等水平，多维度、无偏移的探究CSMD1在增生性瘢痕中的特异性底物及信号转导通路，从而寻找增生性瘢痕靶向治疗的靶点，探索瘢痕治疗新方法。

增生性瘢痕是一类最常见的疾病，表现为成纤维细胞的异常增殖、迁移以及细胞外基质成分的过度沉积。目前致病机制尚未被完全阐明，导致临床上对病理性瘢痕的治疗仍然缺乏有效的手段。.本项目基于全球最大样本量增生性瘢痕GWAS研究，筛选出的与HTS严重程度显著相关的CSMD1基因。验证发现：CSMD1在增生性瘢痕组织和原代细胞中低表达（增生性瘢痕vs正常皮肤，p＜0.01），敲低CSMD1可促进成纤维细胞迁移和FN1分泌。为进一步探究其机制，从其上下游着手：（1）上游通过数据库筛选出调控CSMD1基因的关键microRNA——miR-190a-3p。miR-190a-3p 与CSMD1 3’-非翻译区结合并抑制其表达，过表达/敲低miR-190a-3p促进/抑制成纤维细胞的迁移及FN1分泌，转录组测序结果显示在CSMD1敲低的成纤维细胞中JAK/STAT信号通路被显著激活。（2）CSMD1其可能调控下游TGF-β通路亦是瘢痕形成的重要信号通路。由于TGF-β作用过于广泛，不宜直接干预。因此我们聚焦于一种激活TGF-β 功能的关键分子——血小板反应蛋白-1（TSP-1）。我们发现TSP-1选择性拮抗剂LSKL肽在体外显著抑制增生性瘢痕成纤维细胞的Collagen I和α-SMA mRNA及蛋白表达水平，抑制细胞增殖、迁移及促进凋亡。体内给予LSKL肽能够抑制大鼠鼠尾增生性瘢痕的增生，减少瘢痕厚度和胶原沉积水平及Collagen I及α-SMA蛋白表达。机制探究提示LSKL肽是通过减少PI3K/AKT/mTOR蛋白磷酸化水平，从而导致增生性瘢痕成纤维细胞功能改变。.上述发现miR-190a-3p是增生性瘢痕潜在的诊断、治疗靶点；而LSKL肽作为TSP-1的选择性抑制剂，具有抗瘢痕作用强、副作用小的特点，是一种有临床转化潜能的药物。