Effects of exosomes derived from canine umbilical cord mesenchymal stem cells on canine skin wound

doi:10.3969/j.issn.2095-4344.0959

Abstract

Abstract:

BACKGROUND: Studies have found that mesenchymal stem cell-derived exosomes can inhibit inflammation,
promote skin cell proliferation and angiogenesis, and reduce myofibroblast accumulation. In other words, the injury repair by mesenchymal stem cells is mainly implemented via paracrine mechanism, providing a new method for the treatment of skin injuries. However, there are few studies on the treatment of skin wounds using canine umbilical cord mesenchymal stem cell-derived exosomes.
OBJECTIVE: To investigate the repair effect of umbilical cord mesenchymal stem cell-derived exosomes on canine skin wounds and the underlying mechanisms.
METHODS: Twelve healthy dogs with the same age and growth status were selected. A 4 cm×4 cm square full-thickness incision was made under the medial scapula of the dog back and these 12 dog models were randomly divided into two groups, with 6 rats in each group. The treatment group was injected with 1 mL of saline containing 300 mg/L exosomes, while the model group was injected with the same volume of normal saline as a control. Before and after the modeling, the dog’s mental status and wound healing were observed daily. The blood routine and the wound area were measured regularly and the callus tissues were taken to make pathological observation in the 1st, 2nd and 4th week after modeling.
RESULTS AND CONCLUSION: In the 1st week, there was a marked inflammatory reaction in the model group, and the number of white blood cells was also increased by 30% compared with the baseline data. The inflammatory response in the treatment group was not obvious, and the number of white blood cells and neutrophils was increased slowly as compared with the model group. There was no significant difference in the number of lymphocytes and red blood cells between the two groups. The growth of granulation tissue and wound healing in the treatment group were faster compared with the model group, but no significant differences were detected. These results suggest that canine umbilical cord mesenchymal stem cell-derived exosomes, to some extent, inhibit inflammation and promote skin wound healing.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Umbilical Cord, Mesenchymal Stem Cells, Exosomes, Skin, Stem Cells

CLC Number:

R394.2

Zhan Xiao-shu, Luo Dong-zhang, Wang Bing-yun, Lu Yu, Xian Wei-hang, Lan Yi-qi, Guo Jian-bo, Luo Hui-na, Bai Yin-shan, Ji Hui-qin, Chen Sheng-feng, Chen Zhi-sheng, Liu Can-ying. Effects of exosomes derived from canine umbilical cord mesenchymal stem cells on canine skin wound[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(25): 4021-4027.

Figures/Tables 2

2.1 犬脐带间充质干细胞的培养与鉴定细胞贴壁24 h后进行第1次换液，可见散在的短梭形细胞贴壁，部分增殖形成细胞集落，呈玫瑰花状(图1A)。原代培养7 d后，细胞增殖速度加快，密度明显增加，此时细胞呈长梭形生长(图1B)。培养至第10天时，细胞长满至90%，此时细胞形态均一，活性良好，可进行传代(图1C)。传代后细胞快速增殖，2∶1传代时每一两天可传代1次。由生长曲线图可看出，犬脐带间充质干细胞在P3代时活性最好，增殖最快，随着代数的增加，其增殖速率降低(图2)。P3、P6、P9群体倍增时间分别为23 h、37 h和70 h。免疫荧光检测，犬脐带间充质干细胞的细胞表面CD44呈阳性表达(图3A-C)，CD34呈阴性表达(图3D-F)，符合间充质干细胞特性；将P3代细胞进行成骨分化10 d后形成大量钙结节，用茜素红染色可染成红色(图4B)，成脂分化21 d后形成透亮脂滴，用油红O可染成红色(图4D)。 2.2 犬UC-MSC-EXO的鉴定结果透视电镜下观察，分离和提纯的犬UC-MSC-EXO呈杯托状(图5)，NTA粒径检测显示其大小在30-200 nm，集中在84 nm(图6)。Western Blotting结果显示，犬UC-MSC-EXO表达特异性的外泌体表面蛋白CD81、CD63和CD9(图7)。以上特性表明，分离获得的囊泡体即为外泌体。 2.3 犬UC-MSC-EXO治疗犬皮肤创伤情况犬全层皮肤损伤模型造模后，未见明显红肿热痛现象，饮食和精神状态均正常。在第3天时，模型组伤口处出现较多黄色浓稠炎症产物，而外泌体组伤口则无明显炎症反应；治疗12 d后，两组伤口面积均大幅变小，肉芽组织增生填满伤口，并从四周向中央开始上皮化，但外泌体组较模型组上皮化更为明显；治疗3周后，外泌体组的犬只伤口开始愈合，且伤口较为光滑、平整(图8)。 2.4 犬UC-MSC-EXO治疗后犬血液常规指标的变化从表1可知，造模前两组白细胞总数无差异。造模后3 d 模型组的白细胞数迅速上升，较造模前提高了33%，而外泌体组只升高了10.3%；从第8天开始，模型组的白细胞总数下降，以后一直与外泌体组相近；模型组中性粒细胞数在造模第3天快速升高，而后缓慢降低，而外泌体组在治疗过程中升高较缓慢；两组在治疗过程中淋巴细胞数量和红细胞数量均无显著差异(见表1)。 2.5 犬UC-MSC-EXO对犬皮肤创伤面积和愈合速度的影响从表2可见，从伤口愈合情况来看，在治疗第8和12天时，外泌体组创面面积较模型组小，愈合速度较快，但两组的创面面积和愈合速度无显著差异(P > 0.05)。 2.6 犬UC-MSC-EXO治疗后犬皮肤创伤病理组织学观察从图9可见，第1周时伤口均被结缔组织增生形成的肉芽组织填满，此时增生组织中存在大量纤维细胞，开始形成形状较不规则的单个胶原纤维，由于表皮组织的再生能力远弱于结缔组织，此时还未见表皮层形成。模型组增生组织较外泌体组生长状况差，表面粗糙不平，有较多炎性细胞堆积，并且开始有脂肪细胞出现；第2周时两组伤口表皮均开始生长，均未见角质层及毛囊、皮脂腺、汗腺等皮肤附属结构，但外泌体组伤口再上皮化程度优于模型组；第4周时两组伤口均完全愈合，形成真皮层和表皮层，外泌体组胶原纤维排列成束较模型组更规则，外泌体组可见有汗腺导管再生，两组均未见明显皮脂腺、毛囊再生和角质层形成。"

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