Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice. Methods: The experimental research methods were adopted. The discarded adipose tissue was collected from 3 female patients (aged 10-25 years) who underwent abdominal surgery in the First Affiliated Hospital of Air Force Medical University. ADSCs were extracted from the adipose tissue by collagenase Ⅰ digestion and identified with flow cytometry. Exosomes were extracted from the human ADSCs by differential ultracentrifugation, the morphology of the exosomes was observed by transmission electron microscopy, the particle diameter of the exosomes was detected by nanoparticle tracking analyzer, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin were detected by Western blotting. The human ADSCs exosomes (ADSCs-Exos) and RAW264.7 cells were co-cultured for 12 h, and the uptake of RAW264.7 cells for human ADSCs-Exos was observed. The RAW264.7 cells were divided into phosphate buffer solution (PBS) group stimulated with PBS for suitable time, endotoxin/lipopolysaccharide (LPS) stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group stimulated with LPS for corresponding time, with 3 wells in each group, and the mRNA expressions of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6, and IL-10 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) method. The RAW264.7 cells were divided into PBS group, LPS alone group, and LPS+ADSCs-Exos group, with 3 wells in each group, which were dealt correspondingly for the time screened out in the previous experiment, the mRNA expressions of IL-1β, TNF-α, IL-6, IL-10, trasforming growth factor β (TGF-β,) and vascular endothelial growth factor (VEGF) were detected by real time fluorescence quantitative RT-PCR method, and the protein expressions of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) were detected by Western blotting. Twenty-four 8-week-old male BALB/c mice were divided into PBS group and ADSCs-Exos group according to the random number table, with 12 mice in each group, and a full-thickness skin defect wound with area of 1 cm×1 cm was inflicted on the back of each mouse. Immediately after injury, the wounds of mice in the two groups were dealt correspondingly. On post injury day (PID) 1, the concentration of IL-1β and TNF-α in serum were detected by enzyme-linked immunosorbent assay, and the mRNA expressions of IL-1β, TNF-α, and IL-6 were detected by real time fluorescence quantitative RT-PCR method. On PID 3, 6, 9, 12, and 15, the wound healing was observed and the wound non-healing rate was calculated. On PID 15, the defect length of skin accessory and collagen volume fraction (CVF) were detected by hematoxylin eosin staining and Masson staining, respectively, the CD31 expression and neovascularization were detected by immunohistochemistry, and the ratio of Ki67 positive cells, the ratio of iNOS and Arg1 double positive cells, and the ratio of iNOS positive cells to Arg1 positive cells and their fluorescence intensities were detected by immunofluorescence method. The number of samples in animal experiments was 6. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: At 12 h of culture, the cells exhibited a typical spindle shape, which were verified as ADSCs with flow cytometry. The exosomes with a vesicular structure and particle diameters of 29-178 nm, were positively expressed CD9, CD63, and TSG101 and negatively expressed β-actin. After 12 h of co-culture, the human ADSCs-Exos were endocytosed into the cytoplasm by RAW264.7 cells. The mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group were significantly higher than those in PBS group (with t) values of 39.10, 14.55, 28.80, 4.74, 48.80, 22.97, 13.25, 36.34, 23.12, 18.71, 29.19, 41.08, 11.68, 18.06, 8.54, 43.45, 62.31, 22.52, 21.51, and 37.13, respectively, P<0.01). The stimulation 12 h with significant expressions of all the inflammatory factors was selected as the time point in the following experiment. After stimulation of 12 h, the mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS alone group were significantly higher than those in PBS group (with t values of 44.20, 51.26, 14.71, and 8.54, respectively, P<0.01); the mRNA expressions of IL-1β, TNF-α, and IL-6 of RAW264.7 cells in LPS+ADSCs-Exos group were significantly lower than those in LPS alone group (with t values of 22.89, 25.51, and 8.03, respectively, P<0.01), while the mRNA expressions of IL-10, TGF-β, and VEGF were significantly higher than those in LPS alone group (with t values of 9.89, 13.12, and 7.14, respectively, P<0.01). After stimulation of 12 h, the protein expression of iNOS of RAW264.7 cells in LPS alone group was significantly higher than that in PBS group and LPS+ADSCs-Exos group, respectively (with t values of 11.20 and 5.06, respectively, P<0.05 or P<0.01), and the protein expression of Arg1 was significantly lower than that in LPS+ADSCs-Exos group (t=15.01, P<0.01). On PID 1, the serum concentrations of IL-1β and TNF-α and the mRNA expressions of IL-1β, TNF-α, and IL-6 in wound tissue of mice in ADSCs-Exos group were significantly those in lower than PBS group (with t values of 15.44, 12.24, 9.24, 7.12, and 10.62, respectively, P<0.01). On PID 3, 6, 9, 12, and 15 d, the wound non-healing rates of mice in ADSCs-Exos group were (73.2±4.1)%, (53.8±3.8)%, (42.1±5.1)%, (24.1±2.8)%, and 0, which were significantly lower than (82.5±3.8)%, (71.2±4.6)%, (52.9±4.1)%, (41.5±3.6)%, and (14.8±2.5)% in PBS group, respectively (with t values of 4.77, 8.93, 5.54, 7.63, and 7.59, respectively, P<0.01). On PID 15, the defect length of skin accessory in wounds of mice in PBS group was significantly longer than that in ADSCs-Exos group (t=9.50, P<0.01), and the CVF was significantly lower than that in ADSCs-Exos group (t=9.15, P<0.01). On PID 15, the CD31 expression and the number of new blood vessels (t=12.99, P<0.01), in wound tissue of mice in ADSCs-Exos group were significantly more than those in PBS group, and the ratio of Ki67 positive cells was significantly higher than that in PBS group (t=7.52, P<0.01). On PID 15, the ratio of iNOS and Arg1 double positive cells in wound tissue of mice in PBS group was (12.33±1.97)%, which was significantly higher than (1.78±0.29)% in ADSCs-Exos group (t=13.04, P<0.01), the ratio of iNOS positive cells and the fluorescence intensity of iNOS were obviously higher than those of ADSCs-Exos group, and the ratio of Arg1 positive cells and the fluorescence intensity of Arg1 were obviously lower than those of ADSCs-Exos group. Conclusions: The human ADSCs-Exos can alleviate inflammatory response of mouse RAW264.7 cells, decrease macrophage infiltration and secretion of the pro-inflammatory cytokines, increase the secretion of anti-inflammatory cytokines to promote neovascularization and cell proliferation in full-thickness skin defect wounds of mice, hence accelerating wound healing.
目的： 探讨人脂肪间充质干细胞（ADSC）外泌体对小鼠RAW264.7细胞介导的炎症反应和小鼠全层皮肤缺损创面愈合的影响。 方法： 采用实验研究方法。取2020年6—9月于空军军医大学第一附属医院行腹部手术的3例女性患者（10~25岁）废弃脂肪组织，采用Ⅰ型胶原酶消化法提取ADSC，采用流式细胞术进行鉴定。使用差速超高速离心法提取人ADSC外泌体，采用透射电子显微镜观察形态，纳米颗粒跟踪分析仪检测粒径，蛋白质印迹法检测CD9、CD63、肿瘤易感基因101（TSG101）和β肌动蛋白的蛋白表达。将人ADSC外泌体与RAW264.7细胞共培养12 h后，观察RAW264.7细胞对人ADSC外泌体的吞噬情况。将RAW264.7细胞分为采用磷酸盐缓冲液（PBS）刺激适宜时间的PBS组及内毒素/脂多糖（LPS）刺激相应时间点的LPS刺激2 h组、LPS刺激4 h组、LPS刺激6 h组、LPS刺激12 h组、LPS刺激24 h组，每组3孔，采用实时荧光定量反转录PCR（RT-PCR）法检测白细胞介素1β（IL-1β）、肿瘤坏死因子α（TNF-α）、IL-6及IL-10的mRNA表达。将RAW264.7细胞分为PBS组、单纯LPS组、LPS+ADSC外泌体组，每组3孔，按前一实验筛选的时间进行相应刺激，采用实时荧光定量RT-PCR法检测IL-1β、TNF-α、IL-6、IL-10、转化生长因子β（TGF-β）及血管内皮生长因子（VEGF）mRNA表达，采用蛋白质印迹法检测诱导型一氧化氮合酶（iNOS）、精氨酸酶1（Arg1）的蛋白表达。取24只8周龄雄性BALB/c小鼠，采用随机数字表法分为ADSC外泌体组和PBS组，每组12只，在背部造成1 cm×1 cm的全层皮肤缺损创面。伤后即刻，2组小鼠创面分别进行相应的处理。伤后1 d，采用酶联免疫吸附测定法检测血清中IL-1β和TNF-α的浓度，采用实时荧光定量RT-PCR法检测创面组织IL-1β、TNF-α及IL-6的mRNA表达。伤后3、6、9、12、15 d观察创面愈合情况，并计算创面未愈合率；伤后15 d，行苏木精-伊红染色和Masson染色，分别检测创面皮肤附件缺损长度及胶原容积分数（CVF）；免疫组织化学法检测创面CD31表达及血管新生情况；免疫荧光法检测创面Ki67阳性细胞比、iNOS和Arg1双阳性细胞比、iNOS阳性细胞和Arg1阳性细胞的比值及两者的荧光强度。动物实验中样本数均为6。对数据行重复测量方差分析、单因素方差分析、独立样本t检验。 结果： 培养12 h，细胞呈典型梭形结构，经流式细胞术鉴定为ADSC。外泌体呈囊泡状，粒径29~178 nm，表达CD9、CD63及TSG101而不表达β肌动蛋白。共培养12 h后，人ADSC外泌体成功被RAW264.7细胞吞入细胞质。LPS刺激2 h组、LPS刺激4 h组、LPS刺激6 h组、LPS刺激12 h组、LPS刺激24 h组RAW264.7细胞IL-1β、TNF-α、IL-6、IL-10 mRNA表达均明显高于PBS组（t值分别为39.10、14.55、28.80、4.74，48.80、22.97、13.25、36.34，23.12、18.71、29.19、41.08，11.68、18.06、8.54、43.45，62.31、22.52、21.51、37.13，P<0.01），选择各种炎症因子表达均高表达的刺激12 h作为后续实验时间点。刺激12 h后，单纯LPS组RAW264.7细胞IL-1β、TNF-α、IL-6、IL-10 mRNA表达均明显高于PBS组（t值分别为44.20、51.26、14.71、8.54，P<0.01）；LPS+ADSC外泌体组RAW264.7细胞IL-1β、TNF-α、IL-6 mRNA表达均明显低于单纯LPS组（t值分别为22.89、25.51、8.03，P<0.01），而IL-10、TGF-β和VEGF mRNA表达均明显高于单纯LPS组（t值分别9.89、13.12、7.14，P<0.01）。刺激12 h后，单纯LPS组RAW264.7细胞iNOS的蛋白表达明显高于PBS组和LPS+ADSC外泌体组（t值分别为11.20、5.06，P<0.05或P<0.01），Arg1蛋白表达明显低于LPS+ADSC外泌体组（t=15.01，P<0.01）。伤后1 d，ADSC外泌体组小鼠血清中IL-1β和TNF-α浓度及创面组织中IL-1β、TNF-α、IL-6 mRNA表达均明显低于PBS组（t值分别为15.44、12.24，9.24、7.12、10.62，P<0.01）。伤后3、6、9、12、15 d，ADSC外泌体组小鼠创面未愈合率分别为（73.2±4.1）%、（53.8±3.8）%、（42.1±5.1）%、（24.1±2.8）%、0，均分别明显低于PBS组的（82.5±3.8）%、（71.2±4.6）%、（52.9±4.1）%、（41.5±3.6）%、（14.8±2.5）%（t值分别为4.77、8.93、5.54、7.63、7.59，P<0.01）。伤后15 d，PBS组小鼠创面皮肤附件缺损长度明显长于ADSC外泌体组（t=9.50，P<0.01），CVF明显低于ADSC外泌体组（t=9.15，P<0.01）。伤后15 d，ADSC外泌体组小鼠创面组织CD31阳性表达和新生血管数（t=12.99，P<0.01）明显多于PBS组，Ki67阳性细胞比明显高于PBS组（t=7.52，P<0.01）。伤后15 d，PBS组小鼠创面组织iNOS和Arg1双阳性细胞比为（12.33±1.97）%，明显高于ADSC外泌体组的（1.78±0.29）%（t=13.04，P<0.01），且iNOS荧光强度明显强于ADSC外泌体组，Arg1荧光强度明显强于ADSC外泌体组，iNOS阳性细胞和Arg1阳性细胞的比值明显高于ADSC外泌体组（t=35.16，P<0.01）。 结论： 人ADSC外泌体可以减轻小鼠RAW264.7细胞的炎症反应，在小鼠全层皮肤缺损创面中减少巨噬细胞浸润和促炎性细胞因子分泌，增加抗炎细胞因子分泌，促进新生血管形成，增强创面细胞增殖，加速创面愈合。.