低强度脉冲超声增效脂肪间充质干细胞源性外泌体修复糖尿病小鼠皮肤创面的实验研究

Low intensity pulsed ultrasound enhances adipose mesenchymal stem cells-derived exosomes mediated diabetic wound healing in mice:a experimental study

目的探讨低强度脉冲超声(LIPUS)对脂肪间充质干细胞源性外泌体(ADSCs-Exos)修复糖尿病小鼠皮肤创面的增效作用。方法将ADSCs-Exos与人脐静脉内皮细胞(HUVECs)共培养,评估LIPUS对HUVECs摄取ADSCs-Exos的影响。选取SPF级C57BL/6雄性小鼠36只,构建糖尿病小鼠皮肤创面模型,将其随机分为对照组、Exos治疗组及LIPUS+Exos治疗组,每组各12只,应用活体成像观察ADSCs-Exos的分布及作用时间,冰冻切片观察糖尿病小鼠皮肤组织摄取ADSCs-Exos情况;比较各组小鼠治疗后3 d、7 d、10 d、14 d创面闭合率,以及14 d组织学检测情况。结果LIPUS可显著增加HUVECs对ADSCs-Exos的摄取,辐照后ADSCs-Exos的荧光强度(2.98×105)较未辐照者(0.31×105)显著增加,差异有统计学意义(P<0.01)。活体成像观察显示,LIPUS辐照能有效改善皮下注射后ADSCs-Exos的分布,促进其聚集于创面及周围组织,延长外泌体的作用时间;LIPUS+Exos治疗组治疗后1 h、6 h、36 h创面区域ADSCs-Exos的荧光强度分别为24.14×10^(9)、146.93×10^(9)、26.59×10^(9),均高于Exos治疗组(15.23×10^(9)、52.38×10^(9)、10.72×10^(9)),差异均有统计学意义(均P<0.05)。冰冻切片显示,LIPUS+Exos治疗组ADSCs-Exos阳性细胞率为(41.20±1.10)%,高于Exos治疗组[(23.16±1.16)%],差异有统计学意义(P<0.01)。LIPUS+Exos治疗组小鼠治疗后3 d、7 d、10 d、14 d创面闭合率均高于其余两组,差异均有统计学意义(均P<0.05);以LIPUS+Exos治疗组14 d创面闭合率最高,达(98.52±1.14)%。组织学检测显示,与对照组和Exos治疗组比较,LIPUS+Exos治疗组小鼠皮肤新生血管面积最大[(35.12±1.93)mm2],瘢痕长度最短[(1.20±0.20)mm],胶原容积分数最高[(62.35±1.72)%],差异均有统计学意义(均P<0.05)。结论LIPUS能有效促进细胞对ADSCs-Exos的摄取,增强血管新生,加速糖尿病小鼠皮肤创面的修复。

Objective To investigate the synergistic effect of low intensity pulsed ultrasound(LIPUS)on adipose mesenchymal stem cells-derived exosomes(ADSCs-Exos)in repairing diabetic wound healing in mice.Methods ADSCs-Exos was co-cultured with human umbilical vein endothelial cells(HUVECs)in vitro,and the effect of LIPUS on the uptake of ADSCs-Exos by HUVECs was evaluated.In vivo,36 SPF C57BL/6 male mice were selected to construct diabetic skin wound models.The mice were randomly divided into the control group,Exos treatment group and LIPUS+Exos treatment group,with 12 mice in each group.The distribution and duration of ADSCs-Exos using in vivo imaging were observed.ADSCs-Exos uptake in skin tissue were observed by frozen section.The skin wound closure rate of diabetes mice in each group on the 3rd,7th,10th and 14th day after treatment,and the histological evaluation on the 14th day after treatment were compared.Results LIPUS can significantly increase the uptake of ADSCs-Exos by HUVECs,with a fluorescence intensity of 2.98×105 which was higher than that without irradiated(0.31×105),with a statistically significant difference(P<0.01).In vivo imaging observation shows that LIPUS irradiation can effectively improve the distribution of ADSCs-Exos after subcutaneous injection,promote the aggregation of ADSCs-Exos in wounds and surrounding tissues of diabetes mice,and prolong the action time of extracellular vesicles.The fluorescence intensity of ADSCs-Exos in the wound area at 1 h,6 h and 36 h after treatment in the LIPUS+Exos treatment group were 24.14×10^(9),146.93×10^(9),26.59×10^(9),respectively,which were higher than those of the Exos treatment group(15.23×10^(9),52.38×10^(9),10.72×10^(9)),and the differences were statistically significant(all P<0.05).Frozen section showed that the rate of ADSCs-Exos positive cells[(41.20±1.10)%]was higher than that of Exos treatment group[(23.16±1.16)%],and the difference was statistically significant(P<0.01).The skin wound closure rates of the mice in the LIPUS+Exos treatment group on the 3rd,7th,10th,and 14th day after treatment were higher than that of the other two groups,and the differences were statistically significant(all P<0.05).The LIPUS+Exos treatment group had the highest wound closure rate on the 14th day after treatment[(98.52±1.14)%].The histological evaluation showed that compared with the control group and Exos treatment group,the LIPUS+Exos group had the largest skin neovascularization area[(35.12±1.93)mm2],the shortest scar length[(1.20±0.20)mm],and the highest collagen volume fraction[(62.35±1.72%)],and the difference were statistically significant(all P<0.05).Conclusion LIPUS can effectively promote cell uptake of ADSCs-Exos,enhance angiogenesis and accelerate the repair of diabetic wound healing in mice.