Preliminary study of promotion effect of adipose derived stem cell-exosomes on fibroblast proliferation derived from granulation tissues

Objective To study of promotion effect of adipose derived stem cells-exosomes (ADSC-Exo) on fibroblast proliferation derived from granulation tissues. Methods 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. Results 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). Conclusion 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. Key words: Exosomes; Fibroblasts; Burns; Cell proliferation; Adipose derived stem cells