Extracellular Vesicles Derived From Human Adipose-Derived Stem Cell Prevent the Formation of Hypertrophic Scar in a Rabbit Model.
Extracellular Vesicles Derived From Human Adipose-Derived Stem Cell Prevent the Formation of Hypertrophic Scar in a Rabbit Model.
复制标题
源自人类脂肪干细胞的细胞外囊泡可防止兔模型中肥厚性疤痕的形成。
DOI：
10.1097/sap.0000000000002357
复制
发表时间：
2020
影响因子：
1.5
通讯作者：
Yi Yang-Yan
中科院分区：
文献类型：
--
作者：
Zhu Yuan-Zheng;Hu Xuan;Zhang Jing;Wang Zhao-Hui;Wu Shu;Yi Yang-Yan
背景：在伤口愈合过程中预防瘢痕形成具有重要的临床意义。大量研究表明，脂肪来源的干细胞培养基富含细胞因子和细胞外囊泡（EVs），可调节基质重塑，防止伤口愈合后瘢痕形成。因此，利用兔疤痕模型，我们试图证明脂肪来源的干细胞培养基中的哪个因子在防止疤痕形成（EVs或细胞因子）中起主要作用，并揭示其潜在机制。方法从健康女性供体皮下脂肪组织中分离人脂肪干细胞（hASCs）。流式细胞术分析三代hASCs表面CD标记物。采用Oil O染色检测hASCs的成脂分化能力。收集3 ~ 5代hASCs培养基，分别分离EV和无EV培养基。利用透射电镜、NanoSight和CD63、TSG101和Alix表面标记物的Western blotting对细胞外囊泡进行了表征。用Western blotting检测无ev培养基中血管内皮生长因子A （VEGFA）、血小板衍生生长因子B （PDGFB）和转化生长因子β1 （tgf - β1）的含量。在16只新西兰兔的双耳腹侧制造了直径为8毫米的伤口。在右耳创面愈合过程中局部注射人脂肪源性干细胞-细胞外囊泡（hASC-EV）或不含ev的培养基共0.1 mL。同时，将等量的磷酸盐缓冲盐水注射到左耳作为对照。术后第28天切除损伤皮肤及周围组织，进行苏木精和伊红（H&E）、马松（Masson）和α-SMA免疫荧光染色。Western blotting检测瘢痕组织和正常皮肤α-SMA蛋白和ⅰ型胶原蛋白的表达。结果hacs高表达49d、CD90、CD105和CD73，不表达CD34和CD45。在成脂诱导培养基下，hASCs分化为脂肪细胞。透射电镜下，hasc - ev为圆形双层膜囊泡，约95%的颗粒尺寸在50 ~ 200 nm之间。hasc - ev表达与ev相同的表面标志物，包括CD63、TSG101和Alix， VEGFA、PDGFB和tgf - β1表达较少。无ev培养基中VEGFA、PDGFB和TGFβ1高表达，而CD63、TSG101和Alix不表达。在体内，hASC-EV治疗可防止术后第28天肥厚性疤痕的形成，并抑制胶原沉积和肌成纤维细胞聚集。然而，与对照组相比，无ev培养基在同一时间点并不能阻止增生性瘢痕的形成，对胶原沉积和肌成纤维细胞聚集的影响也很小。结论我们的研究表明，hASCs与预防性瘢痕形成治疗相关的原因是旁分泌EVs而不是细胞因子。伤口愈合过程中局部注射hasc - ev可有效防止增生性瘢痕形成，这可能具有临床有益的抗瘢痕作用。
BackgroundPreventing scar formation during wound healing has important clinical implications. Numerous studies have indicated that adipose-derived stem cell culture mediums, which are rich in cytokines and extracellular vesicles (EVs), regulate matrix remodeling and prevent scar formation after wound healing. Therefore, using a rabbit scar model, we tried to demonstrate which factor in adipose-derived stem cell culture mediums plays a major role in preventing scar formation (EVs or cytokines), as well as revealing the underlying mechanism.MethodsHuman adipose-derived stem cells (hASCs) were isolated from the subcutaneous adipose tissue of a healthy female donor. The surface CD markers of third-passage hASCs were analyzed by flow cytometry. The adipogenic differentiation capacity of the hASCs was detected using Oil O staining. A cultured medium of third-to five-passage hASCs was collected for EV and EV-free medium isolations. Extracellular vesicles were characterized using transmission electron microscopy, NanoSight, and the Western blotting for surface markers CD63, TSG101, and Alix. The EV-free medium was characterized by Western blotting for vascular endothelial growth factor A (VEGFA), platelet derived growth factor B (PDGFB), and transforming growth factor β 1 (TGFβ1). Eight-millimeter-diameter wounds were created on the ventral side of both ears of 16 New Zealand rabbits. A total of 0.1 mL of the human adipose-derived stem cell–extracellular vesicle (hASC-EV) or EV-free medium was locally injected into wounds made on the right ears during wound healing. Meanwhile, equal amounts of phosphate buffer saline were injected into the left ears as a control. Biopsies of the wounded skin and surrounding tissue were excised on postoperative day 28 and subjected to hematoxylin and eosin (H&E), Masson, and α-SMA immunofluorescence staining. The protein expression of α-SMA and collagen I in both scar tissues and the normal skin were evaluated via Western blotting.ResultsThe hASCs expressed high levels of 49d, CD90, CD105, and CD73 but did not express CD34 or CD45. The hASCs differentiated into adipocytes under an adipogenic induction medium. Under transmission electron microscopy, the hASC-EVs were circular, bilayer membrane vesicles and approximately 95% of the particles were between 50 and 200 nm in size. The hASC-EVs expressed the same surface markers as EVs, including CD63, TSG101, and Alix and displayed little expression of VEGFA, PDGFB, and TGFβ1. The EV-free medium had a high expression of VEGFA, PDGFB, and TGFβ1 but displayed no expression of CD63, TSG101, and Alix. In vivo, the hASC-EV treatment prevented the formation of hypertrophic scars on postoperative day 28 and suppressed collagen deposition and myofibroblast aggregation. However, the EV-free medium did not prevent the formation of hypertrophic scars on the same time point and had little effect on collagen deposition and myofibroblast aggregation when compared with the control group.ConclusionsOur study suggests that hASCs are associated with preventive scar formation therapy because of paracrine EVs rather than cytokines. A local injection of hASC-EVs during wound healing efficiently prevented hypertrophic scar formation, which may have a clinically beneficial antiscarring effect.