研究脂肪干细胞来源的外泌体(ADSC-Exo)能否促进肉芽组织来源的成纤维细胞的增殖。

取内蒙古医科大学第三附属医院术后废弃脂肪组织，分离培养脂肪干细胞(ADSC)，超滤浓缩离心方法收集ADSC-Exo，电子显微镜下观察其形态，Western Blotting测定表面标志物，Nanosight分析仪检测粒径和浓度；取烧伤创面的肉芽组织，分离培养肉芽组织成纤维细胞，进行波形蛋白免疫组织化学染色。成纤维细胞划痕实验设立空白组和ADSC-Exo组，检测ADSC-Exo在培养24、48 h对成纤维细胞的迁移作用。Transwell共培养实验设立空白对照组、ADSC-Exo共培养组和ADSC共培养组，检测ADSC-Exo与ADSC在培养24、48、72、96 h对成纤维细胞的增殖作用。成纤维细胞划痕实验结果比较采用独立样本t检验；Transwell共培养实验结果采用单因素方差分析。

电子显微镜下ADSC-Exo可见囊泡结构，直径在40～100 nm之间，ADSC-Exo标志物CD81、CD63的蛋白表达阳性，Nanosight检测显示其直径峰值聚集于55 nm。成纤维细胞镜下外形呈多角形或长梭形，波形蛋白免疫组织化学染色示细胞质呈棕黄色。培养24 h，ADSC-Exo组划痕迁移面积(46.2±9.8)%与空白组(31.7±8.6)%比较，差异有统计学意义(t＝2.72, P<0.05)；培养48 h，ADSC-Exo组迁移面积(85.5±5.3)%与空白组(71.2±8.9)%比较，差异有统计学意义(t＝3.37, P<0.01)。Transwell共培养结果示培养48 h ADSC共培养组吸光度值(0.37±0.05)与空白对照组(0.29±0.06)比较差异有统计学意义(P<0.05)；培养72 h，ADSC-Exo共培养组吸光度值(0.51±0.05)和ADSC共培养组吸光度值(0.53±0.08)显著高于空白对照组(0.38±0.06)，培养96 h，ADSC-Exo共培养组吸光度值(0.68±0.07)和ADSC共培养组吸光度值(0.72±0.11)显著高于空白对照组(0.54±0.07)，差异均有统计学意义(P值均小于0.05)。

超滤浓缩离心方法能够分离ADSC-Exo；ADSC-Exo可显著促进肉芽组织来源成纤维细胞的增殖和迁移能力。

To study of promotion effect of adipose derived stem cells-exosomes (ADSC-Exo) on fibroblast proliferation derived from granulation tissues.

Waste adipose tissue after operation was collected from Third Affiliated Hospital of Inner Mongolia Medical University, ADSC-Exo were isolated by using ultrafiltration method, morphology of ADSC-Exo was observed by Nanosight and electron microscope, expression of proteins was detected by Western Blotting, particle size and concentration were measured by Nanosight analyzer. Granulation tissue from burn wounds was extracted; immunological cell staining was performed to observe the Vimentin in fibroblasts derived from granulation tissue. The effect of ADSC-Exo migration behavior on fibroblast was verified by scratch adhesion test at the time point of 24 h and 48 h. Blank control, ADSC-Exo and ADSC co-culture group were set in Transwell co-culture experiment, the promotion effect of ADSC and ADSC-Exo on fibroblast was verified by Transwell co-culture system at the time point of 24, 48, 72 and 96 h. The results of fibroblast scratch test were compared by independent sample t test, and the results of Transwell co-culture experiment were analyzed by one-way ANOVA.

Electron microscope analysis showed ADSC-Exo had the diameter of 40-100 nm with characteristic morphology. The expression of CD81 and CD63 on ADSC-Exo was detected by Western Blotting, the peak diameter of ADSC-Exo detected by Nanosight was 55 nm. The shape of fibroblasts was long spindle under the microscope and histochemical staining of Vimentin showed the cytoplasm was brown. The scratch migration area of fibroblasts treated with ADSC-Exo(46.2±9.8)% for 24 h had statistical significance when comparing with blank group (31.7±8.6) % (t=2.72, P<0.05); the scratch migration area of fibroblasts treated with ADSC-Exo(85.5±5.3)% for 48 h had statistical significance when comparing with blank group(71.2±8.9)%(t=3.37, P<0.01). Transwell co-culture showed that ADSC group (0.37±0.05)was significantly higher in absorbance value than blank control group (0.29±0.06) at 48 h(P<0.05); the absorbance value of ADSC-Exo co-culture group (0.51±0.05) and ADSC co-culture group (0.53±0.08) cultured for 72 h were significantly higher than that of blank control group (0.38±0.06); the absorbance value of ADSC-Exo group (0.68±0.07) and ADSC group (0.72±0.11) cultured for 96 h were significantly higher than that of blank control group (0.54±0.07), the differences were all statistically significant (with P values below 0.05).

ADSC-Exo isolated by ultrafiltration method is stable; ADSC-Exo promotes fibroblasts migration behavior significantly and has promotion effect on fibroblast derived from granulation tissues.