Effects of applying human umbilical cord mesenchymal stem cell exosomes through different pathways to treat full-thickness skin defect wounds in mice
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摘要:
目的 探讨通过创面局部涂抹、创缘皮下注射和尾静脉注射人脐带间充质干细胞（hUCMSC）外泌体治疗小鼠全层皮肤缺损创面的效果，探究应用hUCMSC外泌体治疗创面的最佳给药途径。 方法 该研究为实验研究。从3名25~35岁于内蒙古包钢医院妇产科正常分娩产妇弃用脐带组织中提取hUCMSC外泌体并成功鉴定。选用120只6~8周龄雄性BALB/c小鼠，于其背部制备全层皮肤缺损创面后，按随机数字表法分为对照组（不进行给药处理）、创面局部涂抹组、创缘皮下注射组、尾静脉注射组（每组30只小鼠），分别通过创面局部涂抹、创缘皮下注射、尾静脉注射给予后3组小鼠0.2 mL含200 μg hUCMSC外泌体的磷酸盐缓冲液。伤后7、14、21 d，观察创面的大体情况并计算创面愈合率；采集创面组织，分别通过苏木精-伊红和Masson染色观测病理学变化和胶原纤维情况，行CD31免疫组织化学染色观测新生微血管数量，采用酶联免疫吸附测定法检测肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）的含量。各组各时间点样本数均为10。 结果 伤后7、14、21 d，4组小鼠创面均逐步愈合，其中创缘皮下注射组小鼠创面愈合情况最佳；3个给药组小鼠创面愈合率均显著高于对照组（ P<0.05），创缘皮下注射组及尾静脉注射组小鼠创面愈合率均显著高于创面局部涂抹组（ P<0.05），创缘皮下注射组小鼠创面愈合率均显著高于尾静脉注射组（ P<0.05）。伤后7、14、21 d，与对照组比较，3个给药组小鼠创面组织生长、上皮化的速度显著加快，创面胶原纤维生成数量更多且排列更整齐。伤后7、14、21 d，在每200倍视野下，创面局部涂抹组小鼠创面组织新生微血管数量分别为（24.1±2.5）、（50.7±4.1）、（44.2±2.3）根，创缘皮下注射组小鼠创面组织新生微血管数量分别为（32.2±2.9）、（67.5±4.9）、（53.6±3.7）根，尾静脉注射组小鼠创面组织新生微血管数量分别为（27.8±2.4）、（59.1±3.7）、（49.6±2.6）根，均显著多于对照组的（20.6±1.7）、（46.7±3.4）、（40.9±2.8）根（ P<0.05）；创缘皮下注射组及尾静脉注射组小鼠创面组织中新生微血管数量均显著多于创面局部涂抹组（ P<0.05）；创缘皮下注射组小鼠创面组织中新生微血管数量均显著多于尾静脉注射组（ P<0.05）。伤后7、14、21 d，3个给药组小鼠创面组织中TNF-α和IL-6的含量均较对照组显著减少（ P<0.05），创缘皮下注射组及尾静脉注射组小鼠创面组织中TNF-α和IL-6的含量均显著少于创面局部涂抹组（ P<0.05），创缘皮下注射组小鼠创面组织中TNF-α和IL-6的含量均显著少于尾静脉注射组（ P<0.05）。 结论 创面局部涂抹、创缘皮下注射及尾静脉注射hUCMSC外泌体均可通过减轻过度炎症反应、促进血管新生发挥促进小鼠全层皮肤缺损创面愈合的作用，其中，创缘皮下注射具有更好的治疗效果，即创缘皮下注射是采用hUCMSC外泌体治疗创面的最佳给药途径。 Abstract:Objective To investigate the effects of human umbilical cord mesenchymal stem cell (hUCMSC) exosomes in the treatment of full-thickness skin defect wounds in mice through local wound application, subcutaneous injection at the wound margin, and tail vein injection, and to explore the optimal administration route of hUCMSC exosomes for wound treatment. Methods This study was an experimental study. hUCMSC exosomes were extracted from the discarded umbilical cord tissue of three normal delivery women aged 25-35 years in the Department of Obstetrics and Gynecology of Baogang Hospital of Inner Mongolia and successfully identified. Totally 120 male BALB/c mice aged 6-8 weeks were selected, and full-thickness skin defect wounds were prepared on the back of them. According to the random number table, the injured mice were divided into control group (without drug administration), local wound application group, wound margin subcutaneous injection group, and tail vein injection group (with 30 mice in each group). Mice in the latter three groups were given 0.2 mL phosphate buffer solution containing 200 μg hUCMSC exosomes by local wound application, subcutaneous injection at the wound margin, and tail vein injection, respectively. On post injury day (PID) 7, 14, and 21, the general condition of the wound was observed, and the wound healing rate was calculated; the wound tissue was collected, the pathological changes and collagen fibers were observed respectively by hematoxylin-eosin staining and Masson staining, the number of new microvessels was observed by CD31 immunohistochemical staining, and the content of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) was detected by enzyme-linked immunosorbent assay. The sample number was 10 in each group at each time point. Results On PID 7, 14, and 21, the wounds of mice in the 4 groups all healed gradually, and the wound healing of the mice in wound margin subcutaneous injection group was the best; the wound healing rates of mice in the three administration groups were significantly higher than those in control group ( P<0.05), the wound healing rates of mice in wound margin subcutaneous injection group and tail vein injection group were significantly higher than those in local wound application group ( P<0.05), and the wound healing rates of mice in wound margin subcutaneous injection group were significantly higher than those in tail vein injection group ( P<0.05). On PID 7, 14, and 21, the growth and epithelialization speed of the wound tissue of mice in the three administration groups were significantly accelerated, and the collagen fibers in the wounds of mice in the three administration groups were larger in number and more neatly arranged in comparison with the control group. On PID 7, 14, and 21, under every 200-fold visual field, the number of new microvessels in the wound tissue of mice in local wound application group was 24.1±2.5, 50.7±4.1, and 44.2±2.3, respectively, the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group was 32.2±2.9, 67.5±4.9, and 53.6±3.7, respectively, and the number of new microvessels in the wound tissue of mice in tail vein injection group was 27.8±2.4, 59.1±3.7, and 49.6±2.6, respectively, which was significantly more than 20.6±1.7, 46.7±3.4, and 40.9±2.8 in control group ( P<0.05); the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group and tail vein injection group was significantly more than that in local wound application group ( P<0.05); the number of new microvessels in the wound tissue of mice in wound margin subcutaneous injection group was significantly more than that in tail vein injection group ( P<0.05). On PID 7, 14, and 21, the content of TNF-α and IL-6 in the wound tissue of mice in the three administration groups was significantly less than that in control group ( P<0.05), the content of TNF-α and IL-6 in the wound tissue of mice in wound margin subcutaneous injection group and tail vein injection group was significantly less than that in local wound application group ( P<0.05), and the content of TNF-α and IL-6 in the wound tissue of mice in wound margin subcutaneous injection group was significantly less than that in tail vein injection group ( P<0.05). Conclusions Local wound application, subcutaneous injection at the wound margin, and tail vein injection of hUCMSC exosomes can all promote the wound healing of full-thickness skin defects in mice through alleviating excessive inflammatory response and promoting angiogenesis. Among them, subcutaneous injection at the wound margin has a better therapeutic effect, indicating subcutaneous injection at the wound margin is the optimal administration route for hUCMSC exosomes in wound treatment. -
1 人脐带间充质干细胞外泌体的形态与粒径。1A.外泌体呈现大小均一的茶托状 透射电子显微镜×20 000；1B.外泌体粒径为30~150 nm
注：图1B为横坐标经过lg处理后生成的图
2 4组全层皮肤缺损小鼠伤后各时间点创面愈合情况。2A、2B、2C.分别为对照组伤后7、14、21 d创面，面积逐渐缩小；2D、2E、2F.分别为创面局部涂抹组伤后7、14、21 d创面，面积分别较图2A、2B、2C有所减小；2G、2H、2I.分别为创缘皮下注射组伤后7、14、21 d创面，面积分别较图2A、2B、2C明显减小，且外观与毛发生长更佳；2J、2K、2L.分别为尾静脉注射组伤后7、14、21 d创面，面积分别较图2A、2B、2C有所减小
注：对照组小鼠未接受给药处理；对其余3组小鼠均给予人脐带间充质干细胞外泌体，组名指示相应给药方式
3 4组全层皮肤缺损小鼠伤后各时间点创面组织中胶原纤维情况 Masson×40。3A、3B、3C.分别为对照组伤后7、14、21 d创面，胶原纤维数量少且排列均紊乱；3D、3E、3F.分别为创面局部涂抹组伤后7、14、21 d创面，图3D中胶原纤维情况与图3A相近，图3E、3F中胶原纤维数量分别较图3B、3C略增加但排列也较紊乱；3G、3H、3I.分别为创缘皮下注射组伤后7、14、21 d创面，胶原纤维数量分别明显多于图3A、3B、3C，排列更加整齐；3J、3K、3L.分别为尾静脉注射组伤后7、14、21 d创面，胶原纤维数量分别多于图3A、3B、3C，排列相对整齐
注：对照组小鼠未接受给药处理；对其余3组小鼠均给予人脐带间充质干细胞外泌体，组名指示相应给药方式；胶原纤维呈蓝色
4 4组全层皮肤缺损小鼠伤后各时间点创面组织中新生微血管情况 二氨基联苯胺-苏木精×200。4A、4B、4C.分别为对照组伤后7、14、21 d创面，新生微血管先增多后有一定减少；4D、4E、4F.分别为创面局部涂抹组伤后7、14、21 d创面，新生微血管数量分别较图4A、4B、4C增加；4G、4H、4I.分别为创缘皮下注射组伤后7、14、21 d创面，新生微血管数量分别较图4A、4B、4C明显增加；4J、4K、4L.分别为尾静脉注射组伤后7、14、21 d创面，新生微血管数量分别较图4A、4B、4C增加
注：对照组小鼠未接受给药处理；对其余3组小鼠均给予人脐带间充质干细胞外泌体，组名指示相应给药方式；新生微血管呈棕褐色
表1 4组全层皮肤缺损小鼠伤后各时间点创面愈合率比较（%，
表1. Comparison of wound healing rates of 4 groups of mice with full-thickness skin defects at each time point after injury
组别 鼠数（只） 7 d 14 d 21 d 创面局部涂抹组 10 26.1±4.3 45.7±8.3 85.4±5.0 创缘皮下注射组 10 36.0±5.4 66.8±4.1 99.7±3.4 尾静脉注射组 10 31.6±6.6 59.4±9.4 92.5±4.2 对照组 10 17.3±2.5 40.4±10.6 80.2±5.6 P 1值 <0.001 0.047 <0.001 P 2值 <0.001 <0.001 <0.001 P 3值 <0.001 <0.001 <0.001 P 4值 <0.001 <0.001 <0.001 P 5值 <0.001 <0.001 <0.001 P 6值 0.002 0.003 <0.001 注：对照组小鼠未接受给药处理；对其余3组小鼠均给予人脐带间充质干细胞外泌体，组名指示相应给药方式；时间因素主效应， F=3 051.01, P<0.001；处理因素主效应， F=179.96, P<0.001；两者交互作用， F=6.56, P<0.001； P 1值、 P 2值、 P 3值分别为创面局部涂抹组、创缘皮下注射组、尾静脉注射组与对照组各时间点比较所得； P 4值、 P 5值分别为创缘皮下注射组、尾静脉注射组与创面局部涂抹组各时间点比较所得； P 6值为创缘皮下注射组与尾静脉注射组各时间点比较所得 
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表2 4组全层皮肤缺损小鼠伤后各时间点创面组织中新生微血管数量比较（根，
表2. Comparison of the number of new microvessels in the wound tissue of 4 groups of mice with full-thickness skin defects at each time point after injury
组别 鼠数（只） 7 d 14 d 21 d 创面局部涂抹组 30 24.1±2.5 50.7±4.1 44.2±2.3 创缘皮下注射组 30 32.2±2.9 67.5±4.9 53.6±3.7 尾静脉注射组 30 27.8±2.4 59.1±3.7 49.6±2.6 对照组 30 20.6±1.7 46.7±3.4 40.9±2.8 P 1值 <0.001 0.001 <0.001 P 2值 <0.001 <0.001 <0.001 P 3值 <0.001 <0.001 <0.001 P 4值 <0.001 <0.001 <0.001 P 5值 <0.001 <0.001 <0.001 P 6值 <0.001 <0.001 <0.001 注：对照组小鼠未接受给药处理；对其余3组小鼠均给予人脐带间充质干细胞外泌体，组名指示相应给药方式；各组各时间点样本数为10；于200倍视野下计数新生微血管；时间因素主效应， F=2 889.44， P<0.001；处理因素主效应， F=355.25， P<0.001；两者交互作用， F=16.90， P<0.001； P 1值、 P 2值、 P 3值分别为创面局部涂抹组、创缘皮下注射组、尾静脉注射组与对照组各时间点比较所得； P 4值、 P 5值分别为创缘皮下注射组、尾静脉注射组与创面局部涂抹组各时间点比较所得； P 6值为创缘皮下注射组与尾静脉注射组各时间点比较所得
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表3 4组全层皮肤缺损小鼠伤后各时间点创面组织中TNF-α与IL-6的含量比较（ng/L，
表3. Comparison of the content of TNF-α and IL-6 in the wound tissue of 4 groups of mice with full-thickness skin defects at each time point after injury
组别 鼠数（只） TNF-α IL-6 7 d 14 d 21 d 7 d 14 d 21 d 创面局部涂抹组 30 168±15 108±13 86±10 295±32 152±14 133±12 创缘皮下注射组 30 142±13 84±8 70±9 248±25 126±18 115±10 尾静脉注射组 30 156±15 92±13 78±9 278±22 140±15 124±15 对照组 30 205±15 143±15 101±10 332±30 162±19 142±16 P 1值 <0.001 <0.001 <0.001 <0.001 0.042 0.019 P 2值 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 P 3值 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 P 4值 <0.001 <0.001 0.001 <0.001 <0.001 <0.001 P 5值 0.004 <0.001 0.005 0.044 0.018 0.022 P 6值 <0.001 0.037 <0.001 <0.001 0.005 0.020 注：对照组小鼠未接受给药处理；对其余3组小鼠均给予人脐带间充质干细胞外泌体，组名指示相应给药方式；各组各时间点样本数为10；肿瘤坏死因子α（TNF-α）与白细胞介素6（IL-6）的时间因素主效应， F值分别为1 477.78、2 419.30， P值均<0.001；处理因素主效应， F值分别为282.78、73.30， P值均<0.001；两者交互作用， F值分别为17.30、13.30， P值均<0.001； P 1值、 P 2值、 P 3值分别为创面局部涂抹组、创缘皮下注射组、尾静脉注射组与对照组各指标各时间点比较所得； P 4值、 P 5值分别为创缘皮下注射组、尾静脉注射组与创面局部涂抹组各指标各时间点比较所得； P 6值为创缘皮下注射组与尾静脉注射组各指标各时间点比较所得
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