TNFSF13/HSPG信号通路在NK细胞免疫调控增生性瘢痕中的作用机制研究

Hypertrophic scar (HS) is an abnormal repair of local tissue caused by deep burns and severe trauma which is the difficulty of plastic surgery research. Our study confirmed that natural killer cells (NK) play a key role in the immune regulation of wound repair. Recent studies have reported that tumor necrosis factor superfamily 13 (TNFSF13) was involved in the immune regulation of wound repair, and its weak expression can reduce the formation of fibrous scars. We found that the expression of TNFSF13 was decreased significantly after knocking out NK cells in Hypertrophic scar animals, and scar formation was attenuated significantly. Based on this, we speculate that TNFSF13 may participate in immune regulation of NK cells in the formation of HS, and it is of great significance to further study its regulatory mechanism. We found that Heparan sulfate proteoglycan (HSPG) receptor may be a target for TNFSF13 action in HS tissues while using the protein chip screening. In addition, HSPG has the effect of enhancing the recruitment of inflammatory cells during wound repair and healing. Based on above, this project intends to adopt the methods of molecular biology and drug intervention through human Hypertrophic scar tissue, cells in vitro and animal models in vivo as the research subject, to explores the role and molecular mechanism of TNFSF13/HSPG signaling pathway in NK cells immune regulation of HS, and provides a theoretical basis for immunosuppressive the formation of Hypertrophic scar with NK cells as a new target.

增生性瘢痕(HS)是皮肤深度烧伤、严重创伤后致局部组织的异常修复，是整形外科研究的难点。我们研究证实自然杀伤细胞（NK）在创面修复免疫调控中起关键作用。最新研究报道肿瘤坏死因子超家族13（TNFSF13）参与创面修复的免疫调控，其表达减弱使纤维瘢痕的形成减少。我们实验发现HS动物敲除NK后，瘢痕组织中TNFSF13的表达显著下降，瘢痕缩小显著。因此我们推测TNFSF13可能参与NK免疫调控HS的形成，深入研究其调控机制具有重要意义。利用蛋白芯片筛选我们发现硫酸乙酰肝素蛋白聚糖(HSPG)受体可能是TNFSF13作用的靶点。结合文献报道HSPG在创面修复中具有增强招募炎症细胞的作用。基于此，本项目以人HS组织，体外细胞和体内动物模型为实验对象，采用分子生物学和药物干预等手段，探索TNFSF13/HSPG信号通路在NK免疫调控HS中的作用及分子机制，为以NK为新靶标抑制HS的形成提供理论依据。

背景：病理性瘢痕是成纤维细胞的过度增殖、细胞外基质中胶原的过度沉积以及各种免疫细胞的反应增加所致。瘢痕具体形成机制尚不清楚。.主要研究内容：收集瘢痕疙瘩、正常皮肤组织标本，分别采用NAD/NADH荧光定量法和QPCR法来检测细胞中NAD、SIRT1的表达水平。应用基因干扰及过表达技术包装病毒转染人皮肤成纤维细胞（human skin fibroblast, HSF），分别进行细胞增殖、迁移、及细胞凋亡和细胞周期检测，用Western blot检测NF-κB、TNF-β、α- SMA的表达量，分析SIRT1细胞学功能。通过生信分析，筛选出参与细胞迁移的同时又调控SIRT1基因的miRNA。HSF细胞中，用Transwell、Western blot，最终选取hsa-miR-138-5p。为了验证miR138-5p能否结合SIRT1靶位点从而影响SIRT1，我们用双荧光素酶检测了miR138-5p与SIRT1的结合。为了研究MSC对瘢痕疙瘩组织SIRT1基因的影响，检测经miR138-5p修饰的外泌体对HSF细胞增殖、迁移、凋亡及细胞周期的影响，用Western blot检测经miR138-5p修饰的外泌体对NF-κB、TNF-β、α-SMA的影响。.重要结果和数据：我们证实NAD、SIRT1在瘢痕疙瘩成纤维细胞中的表达量高于其在正常皮肤成纤维细胞中的表达量。另外，Visfatin能增加细胞内和细胞外NAD水平。转染Sh-SIRT1（KD）慢病毒的HSF细胞增殖、迁移能力较NC（KD）明显减弱（P<0.05），细胞周期明显延长，细胞凋亡增加；而转染过表达SIRT1（OE）慢病毒的HSF细胞增殖、迁移较NC（KD）明显增强（P<0.05），细胞周期明显缩短，细胞凋亡减少。双荧光素酶检测结果显示miR138-5p可能直接结合SIRT1-3’UTR靶位点。miR138-5p可以通过电穿孔的方式进入外泌体内，并随外泌体一起进入HSF细胞内。经miR138-5p修饰的外泌体孵育后的HSF细胞较对照组增殖、迁移能力下降，细胞周期延长，细胞凋亡增加；而经miR138-5p抑制剂修饰的外泌体孵育后的HSF细胞较对照组增殖、迁移能力明显增强，细胞周期缩短，细胞凋亡减少。.科学意义：通过我们的研究发现瘢痕的发生发展机制，可以早期进行积极预防，利用干细胞外泌体预防、治疗瘢痕。