人脐带间充质干细胞来源外泌体对小鼠压疮的治疗作用及机制

Therapeutic effect and mechanism of human umbilical cord mesenchymal stem cell-derived exosome on wound healing of pressure ulcers in mice

目的探索人脐带间充质干细胞(huc MSC)来源外泌体(huc MSC-Exo)对小鼠4期压疮创面愈合的作用及其机制。方法原代分离、培养人脐带间充质干细胞,取第5代(P5代)细胞培养上清,通过差速离心法提取huc MSC-Exo。将27只成功制备4期压疮模型的BALB/c小鼠随机分为huc MSC-Exo治疗组(创周皮下注射100μg溶解于100μL PBS中的huc MSC-Exo)、PBS对照组(创周皮下注射100μL PBS)及模型对照组(未给予huc MSC-Exo和PBS处理)。治疗后评估压疮创面愈合情况,于0、3、7、10、14d拍照并计算创面愈合率;取治疗后7、14 d皮肤样本行H-E染色和Masson染色观察创面病理变化;取治疗后7 d皮肤样本行免疫组织化学染色检测与瘢痕形成相关的α平滑肌肌动蛋白(α-SMA)表达;取治疗后7、14 d血清检测氧化应激指标超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量的动态变化。结果huc MSC-Exo治疗组较PBS对照组及模型对照组创面愈合速度加快,瘢痕形成减少,创面愈合率升高。组织病理学结果显示,与PBS对照组及模型对照组比较,huc MSC-Exo治疗组创面损伤程度、炎性浸润情况明显减轻,胶原沉积量增多,胶原纤维排列分布有序。免疫组织化学染色结果显示,huc MSC-Exo治疗组α-SMA蛋白表达少于PBS对照组及模型对照组。血清生物化学检测结果示,huc MSC-Exo治疗组SOD活性在治疗后7 d高于PBS对照组及模型对照组(P均<0.05),到14 d接近正常水平;MDA含量在7 d和14 d均低于PBS对照组及模型对照组(P均<0.05)。结论皮下注射huc MSC-Exo可促进小鼠4期压疮创面愈合,减少瘢痕形成,其机制可能与huc MSC-Exo改善体内氧化应激水平有关。

Objective To explore the effect and mechanism of human umbilical cord mesenchymal stem cell(huc MSC)-derived exosome(huc MSC-Exo)on wound healing of stage 4 pressure ulcers in mice.Methods huc MSCs were isolated and cultured in primary culture.huc MSC-Exos were extracted from the supernatant of huc MSCs at passage 5(P5)through differential centrifugation.Twenty-seven BALB/c mice with stage 4 pressure ulcers were randomly divided into huc MSC-Exo group(injected subcutaneously with 100μg huc MSC-Exo dissolved in 100μL phosphate-buffered saline[PBS]),PBS group(injected subcutaneously with 100μL PBS),and model group(without injection of huc MSC-Exo or PBS).The wound healing of pressure ulcers was assessed after treatment,and the wound healing rate was estimated on day 0,3,7,10,and 14.Hematoxylin-eosin(H-E)staining and Masson staining were used to observe the pathological changes of the wound on day 7 and 14 after treatment.The expression ofα-smooth muscle actin(α-SMA)protein related to scar formation was detected by immunohistochemical staining with skin samples on day 7 after treatment.Serum samples were obtained on day 7 and 14 after treatment to detect the dynamic changes of superoxide dismutase(SOD)activity and malondialdehyde(MDA)content.Results Compared with the PBS and model groups,the huc MSC-Exo group had much shorter wound healing time,less scar formation,and higher wound healing rate.Histopathology examination showed that,compared with the PBS and model groups,the wound injury and inflammatory infiltration in the huc MSC-Exo group were significantly decreased,collagen deposition was greatly boosted,and collagen fibers were orderly arranged and distributed.Immunohistochemical staining showed that the expression ofα-SMA protein in the huc MSC-Exo group was lower than those in the PBS and model groups.The results of serum biochemical test showed that SOD activity in the huc MSC-Exo group was higher than those in the PBS and model groups(both P<0.05)on day 7 after treatment,approaching the normal level on day 14;MDA content was lower in the huc MSC-Exo group than the PBS and model groups on day 7 and 14(all P<0.05).Conclusion Subcutaneous injection of huc MSC-Exo can promote wound healing and minimize scar formation in mice with stage 4 pressure ulcers.The mechanism may be related to the improvement of in vivo oxidative stress levels by huc MSC-Exo.