脂肪干细胞外泌体miR-192-5p靶向抑制IL-17RA/LC3通路拮抗增生性瘢痕形成的机制研究

Hypertrophic scar (HS), as a serious skin fibrotic disease, has always been a major problem for clinical treatment. The reports and our previous research confirmed: adipose-derived stem cells (ADSCs) inhibited HS formation, but the mechanism remains unknown. The new research demonstrated exosome played a pivotal role in alleviating tissue fibrosis for stem cell, and recently we found miR-192-5p was overexpressed in ADSCs-derived exosome(ADSC-Exo); and there were the binding domains of miR-192-5p in the sequence of IL-17RA mRNA 3′-UTR, suggesting that miR-192-5p might target IL-17RA to inhibit its expression, and IL-17A/IL-17RA was overexpressed in HS and IL-17A could up-regulate the expression of LC3 in HS-derived fibroblasts to facilitate the skin fibrosis. Therefore, we presented out the following hypothesis: ADSC-Exo-miR-192-5p combined with IL-17RA to regulate the level of LC3, which lead to the hypertrophic scar formation. Based on this, Firstly, we will verify the effect of ADSC-Exo on HS formation; Furthermore, it is necessary to explore the expression of miR-192-5p and the key molecular of IL-17A/IL-17RA signaling pathway in HS and observe the expression of IL-17RA, LC3 pathway and the fibrotic molecules by the exogenous interference of exosome miR-192-5p; Moreover,we use IL-17RA inhibitors/siRNA to validate the aforementioned effect in both cellular level and rabbit ear HS model, and elucidate the role of IL-17RA/LC3 signaling pathway in HS formation. This study will reveal the potential mechanism of the anti-fibrosis effect of ADSCs in HS formation and provide a new strategy for the treatment of hypertrophic scar.

增生性瘢痕是临床亟需解决的皮肤纤维化难题。前期证实脂肪干细胞抑制瘢痕形成，但机制不明。最新研究表明外泌体是干细胞减轻纤维化的关键途径，近期预实验发现脂肪干细胞外泌体高表达miR-192-5p，其靶点可能为IL-17RA，之后检测到IL-17A/IL-17RA在瘢痕中表达增高，且IL-17A上调成纤维细胞中LC3的表达促进瘢痕的纤维化。因此提出假说:脂肪干细胞外泌体miR-192-5p靶向IL-17RA调控LC3通路作用于瘢痕的形成。据此,本课题拟证实脂肪干细胞外泌体对瘢痕的作用；探讨miR-192-5p及IL-17RA通路分子在瘢痕中的表达及外源干预外泌体miR-192-5p对IL-17RA，LC3通路及纤维化因子的影响；并用IL-17RA siRNA对细胞及动物验证，明确IL-17RA/LC3通路在瘢痕中的作用。该课题有望揭示脂肪干细胞抑制瘢痕形成的潜在分子机制，为临床治疗提供新途径。

增生性瘢痕是创面过度愈合的结果，作为一种严重的皮肤纤维化疾病，常伴随外形的毁损和不同程度的功能障碍，是组织修复领域亟待解决的难题。文献及临床实验提示：脂肪干细胞在瘢痕治疗中的应用日益凸显，旁分泌功能是关键，而外泌体作为干细胞旁分泌功能的重要效应载体，被认为与干细胞有相似的功能。本课题阐明了脂肪干细胞外泌体对瘢痕纤维化的调控作用，证实了mir-192-5p是外泌体发挥作用的关键分子，确认了mir-192-5p与IL-17RA的靶向关系，揭示了IL-17A/IL-17RA对瘢痕成纤维细胞自噬通路的影响，从而验证了此前提出的科学假说：脂肪干细胞外泌体mir-192-5p靶向IL-17RA/LC3通路拮抗增生性瘢痕的形成。具体实验结果表明脂肪干细胞外泌体抑制瘢痕成纤维细胞的增殖、迁移、收缩及纤维化因子的表达；促进小鼠创面的愈合，减少胶原的表达及沉积，抑制成纤维细胞向肌成纤维细胞的转分化；兔耳瘢痕模型中也发现其改善瘢痕的纤维化及胶原的排列。接着通过测序分析及qPCR检测发现脂肪干细胞外泌体高表达mir-192-5p；共培养，外泌体分泌抑制剂GW4869及荧光标记实验证实了mir-192-5p是外泌体发挥拮抗瘢痕纤维化作用的关键所在；抑制外泌体mir-192-5p的表达促进瘢痕成纤维细胞生物学功能的恢复及上调纤维化因子的表达，佐证了其重要性。进而通过序列对比分析，荧光素酶报告实验及过表达抑制mir-192-5p证实了其与IL-17RA的靶向关系，同时检测了IL-17RA在瘢痕中的表达及干扰IL-17RA对瘢痕成纤维细胞及小鼠创面的作用，并验证了IL-17A/IL-17RA对自噬通路的影响。此外，也完善了外泌体及源性mir-192-5p对IL-17RA及自噬通路关键分子表达的作用。通过上述实验，有望揭示脂肪干细胞抑制瘢痕纤维化的潜在分子生物学机制，为临床提供新的理论基础和治疗前景。