羊膜上皮干细胞外泌体通过miR-let-7/TfR1途径拮抗增生性瘢痕的机制研究

Hypertrophic scar (HS), a serious skin fibrotic disease, has always been a challenging issue in clinical medicine. Reports and our previous researches showed that HS has a common characteristic of high intracellular iron content resulted from the iron metabolism disorder, indicating that iron overload may be an important risk factor in HS. Recently, we found that amniotic epithelial stem cell-derived exosomal miR-let-7 not only inhibited HS formation, but also affected genes expression of iron metabolism on fibroblasts of HS. Based on this observation, we speculate that exosomes derived from human amniotic membrane epithelial stem cells regulate iron metabolism homeostasis of HS-derived fibroblasts by transfer miR-let-7, thereby regulate cell differentiation and antagonize HS fibrosis. However, the exact molecular mechanism remains unclear. Therefore, the current project is aimed to evaluate the roles of miR-let-7 carried within the amniotic epithelial stem cell-derived exosomes, which are found to antagonize HS fibrosis, and to investigate the molecular mechanism of exosomal miR-let-7 regulated iron metabolic homeostasis of HS-derived fibroblasts by in vitro and in vivo models. This research will provide potential molecular target and theoretical evidence for the treatment of HS.

增生性瘢痕（HS）作为严重的皮肤纤维化疾病，是临床医学面临的难题和挑战。文献及我们前期研究证实铁代谢稳态失衡是HS的典型特征，铁过载可能是瘢痕增生的一个重要危险因素。课题组近期研究发现人羊膜上皮干细胞来源外泌体miR-let-7不仅抑制瘢痕形成，而且能够调控HS成纤维细胞铁代谢功能。据此我们提出假设：人羊膜上皮干细胞外泌体通过miR-let-7调控HS成纤维细胞铁代谢功能，从而影响HS成纤维细胞分化及胞外基质重塑，最终拮抗瘢痕纤维化，但其具体调控机制尚需进一步验证。因此，本课题拟采用外泌体XMIRXPress、miR过表达/干扰、双荧光素酶报告基因、CRISPR/Cas9介导的靶基因敲除/敲入、小动物活体成像技术和生物信息学等方法，通过在体和离体模型探讨并验证人羊膜上皮干细胞外泌体通过miR-let-7调控HS成纤维细胞铁代谢功能，拮抗瘢痕纤维化的分子机制，为HS的治疗提供新靶点及思路。

增生性瘢痕（HS）作为烧、创伤创面愈合后不可避免的附属产物，是临床医学面临的难题和挑战。文献及我们前期研究证实铁代谢稳态失衡是HS形成的一个重要危险因素。课题组近期研究发现人羊膜上皮干细胞来源外泌体不仅能够抑制HS形成，而且能够调控HS成纤维细胞铁代谢功能。既往研究表明外泌体是细胞靶向转运miRNAs的主要载体之一。据此，本课题基于铁代谢深入研究了人羊膜上皮干细胞来源外泌体及其miRNAs拮抗瘢痕增生的作用及机制。首先，本研究明确了人羊膜上皮干细胞外泌体能够抑制瘢痕增生。其次，通过RNA-Seq高通量测序，候选出了羊膜上皮干细胞外泌体中调控铁代谢和皮肤纤维化相关的一系列miRNAs（miR-34, miR-let-7, miR-21, miR-31，miR-7），经过验证，最后筛选出miR-let-7。应用生物信息学分析得到了miR-let-7与铁摄取密切相关的靶基因TfR1, DMT1，通过双荧光素酶报告基因在细胞水平上验证得到DMT1是miR-let-7的靶基因。最后，利用siRNA技术过表达及沉默DMT1，验证DMT1在调控铁代谢和瘢痕增生中的功能，最终阐明人羊膜上皮干细胞拮抗瘢痕纤维化的作用机理，即人羊膜上皮干细胞通过外泌体运输释放miR-let-7，靶向沉默铁摄取相关基因DMT1的表达，降低HS成纤维细胞中铁离子表达水平，进而抑制细胞的增殖和胞外基质的分泌，最终拮抗瘢痕纤维化。该课题为瘢痕的防治提供了潜在的作用靶点及理论依据，为干细胞外泌体在整形美容领域的临床转化应用具有积极的推动作用。