摘要：

目的：探讨人唾液外泌体（saliva-derived exosomes，S-Exos）对大鼠皮肤损伤修复的影响。方法：收集健康成年人唾液，分离并鉴定唾液外泌体。将18只 SD大鼠随机分为对照组、唾液处理组和外泌体处理组。通过皮肤打孔法制备大鼠皮肤全层缺损模型，各组在建模后48 h时于创面边缘分4点进行皮下注射给药100 μL。在建模后第0、4、8、12、14天时观察各组大鼠创面愈合情况，计算伤口愈合率。于建模后第14和第35天处死大鼠，取创面皮肤组织，采用HE染色观察皮肤组织形态表现。结果：在透射电镜观察下，S-Exos呈杯状，粒径大小约为70 nm，蛋白质免疫印迹（Western blotting）结果显示，外泌体特异性标志物CD63和CD9均为阳性。动物实验表明，S-Exos可以促进大鼠皮肤伤口面积收缩。在建模后第8天，外泌体处理组的愈合率显著高于对照组和唾液处理组（P<0.01）。建模后第14天，外泌体处理组可见创面新生组织中成纤维细胞和炎症细胞的细胞数目及血管生成增多，建模后第35天可见胶原纤维有序平行排列，并有个别毛囊生成。结论：人唾液外泌体能够促进大鼠皮肤全层损伤的愈合，调控损伤修复过程中的炎症反应，诱导成纤维细胞的增殖，有助于损伤部位皮肤组织恢复正常的组织形态。

关键词: 皮肤损伤, 唾液, 外泌体, 损伤修复

Abstract:

Objective: To investigate the effect of human saliva-derived exosomes (S-Exos) on the healing of skin injury in rats. Methods: Saliva samples of healthy adults were collected and S-Exos were isolated and identified. 18 Sprague Dawley (SD) rats were randomly divided into control group, saliva group and S-Exos group respectively. Full-thickness excisional skin wounds were created with a skin puncher at the dorsum of each rat, and a full-thickness dermal defect model was established. 100 μL ingredients of S-Exos were injected subcutaneously at four sites of the wound margin at 48 hours after model establishment. The healing situation of wounds was observed at 0, 4, 8, 12, and 14 days after model establishment, and the wound healing rate was calculated. The rats were sacrificed at 14 and 35 days after model establishment and skin samples in defect area were harvested. HE staining was used to observe the histological morphology of skin. Results: S-Exos exhibited cup-shaped morphologies, the particle size was about 70 nm under transmission electron microscope (TEM). Western blotting results showed CD63 and CD9 as specific markers of exosomes were positively expressed in S-Exos. S-Exos could promote the contraction of rat skin wound area. At 8 days after model establishment, the wound healing rate of S-Exos group was significantly higher than that of the control and salivary groups (P<0.01). The skin histopathological evaluation showed that the number of fibroblasts and inflammatory cells and microvessels in S-Exos group increased at 14 days after model establishment. Collagen fibers were orderly and arranged in parallel and individual hair follicles were formed at 35 days after model establishment. Conclusion: Human S-Exos could promote wound healing of full-thickness skin defect injury in rats, modulate inflammatory process during wound healing inducing fibroblasts proliferation, and benefit healing to restore to normal histomorohology.

Key words: skin injury, saliva, exosomes, damage repair