目的

研究负载人脂肪干细胞来源外泌体的甲基丙烯酰化明胶（GelMA）水凝胶对人皮肤成纤维细胞增殖和迁移的作用。

方法

分离和培养人脂肪干细胞，检测其表面分子标志物的表达。采用超速离心法提取人脂肪干细胞来源外泌体，应用扫描电镜、纳米流式检测技术进行鉴定。 培养人皮肤成纤维细胞，采用免疫荧光检测细胞对外泌体的内化；分别加入浓度为1×10⁷/ml、 1×10⁸/ml 及1×10⁹/ml 的外泌体，检测外泌体对人皮肤成纤维细胞增殖和迁移的影响。 制备浓度为5%、 10%、 15%的GelMA 水凝胶，扫描电镜下观察GelMA 水凝胶微观结构并进行孔径分析，检测水凝胶在体外的降解速度；采用ELISA 法检测外泌体在水凝胶中的缓释速度，然后在水凝胶中共培养人皮肤成纤维细胞，进行活/死细胞染色，检测细胞在水凝胶中的生长及增殖情况。

结果

扫描电镜及纳米流式检测结果显示获取的粒子为外泌体。 荧光标记的人脂肪干细胞来源外泌体能够被人皮肤成纤维细胞摄取进入细胞质及细胞核中。 浓度为1×10⁷/ml、1×10⁸/ml、1×10⁹/ml 的外泌体均可促进人皮肤成纤维细胞的增殖和迁移（F=3.579，P=0.021）；浓度为1×10⁸/ml、 1×10⁹/ml 外泌体对细胞的增殖作用较浓度为1×10⁷/ml 外泌体更强（P=0.048、P=0.005 ），作用24 h 后浓度为1×10⁸/ml 外泌体与1×10⁹/ml 外泌体对细胞的增殖作用差异无统计学意义（P=0.091、P=0.083）。 浓度为5%、10%、15%的GelMA 水凝胶体外降解速度随浓度增加而减慢，浓度为5%的GelMA 水凝胶能更好地缓慢持续释放外泌体，在5%的GelMA 水凝胶中加入人皮肤成纤维细胞培养后，细胞在水凝胶中生长及增殖良好。

结论

人脂肪干细胞来源外泌体被人皮肤成纤维细胞内化后可以促进其增殖和迁移。 浓度为5%负载人脂肪干细胞外泌体（1×10⁸/ml）的GelMA 水凝胶可缓慢持续释放外泌体，促进人皮肤成纤维细胞增殖，可作为外泌体的良好生物载体。

Objective

To observe the effect of gelatin methacryloyl hydrogel loaded with exosomes from human adipose-derived stem cells on the proliferation and migration of human skin fibroblasts.

Methods

Human adipose-derived stem cells were isolated and cultured， and the expression of molecular markers on the surface was detected.Human adipose stem cell-derived exosomes were extracted by ultracentrifugation and identified by scanning electron microscopy and Nano-flow cytometry.Human skin fibroblasts were cultured， and immunofluorescence was performed to detect the internalization of the exosomes.The exosomes were added at concentrations of 1×10⁷/ml， 1×10⁸/ml， and 1×10⁹/ml espectively to detect the effects of exosomes on the proliferation and migration of human skin fibroblasts.GelMA hydrogels were prepared at concentrations of 5%，10%，and 15%， and the microstructures of the hydrogels were observed by scanning electron microscopy and pore size was analyzed.The in vitro degradation rate of the hydrogels was measured.ELISA was used to detect the sustained-release rate of exosomes in the hydrogel.Human skin fibroblasts were co-cultured in the hydrogel， and live-dead cell staining was performed to detect the growth and proliferation of cells in the hydrogel.

Results

The electron microscopy and nanofluidic detection showed that the acquired particles were exosomes.Fluorescently labeled human adipose stem cell-derived exosomes could be internalized by human skin fibroblasts into the cytoplasm and nucleus.The concentrations of 1×10⁷/ml，1×10⁸/ml， and 1×10⁹/ml of exosomes all promoted the proliferation and migration of human skin fibroblasts（F=3.579，P=0.021）.For proliferation， the effect of exosomes at concentrations of 1×10⁸/ml and 1×10⁹/ml was more substantial than that of 1×10⁷/ml （P=0.048，P=0.005）.After 24 hours of treatment， there was no statistically significant difference in the effect of exosomes at concentrations of 1×10⁸/ml and 1×10⁹/ml on cell proliferation （P = 0.091， P = 0.083）.The in vitro degradation rate of GelMA hydrogel at concentrations of 5%， 10% and 15% decreased with the increase of concentration， the hydrogel at a concentration of 5% was better in releasing the exosomes in a slow and sustained manner.After culturing human skin fibroblasts in the 5% GelMA hydrogel， the cells grew and proliferated favorably in the hydrogel.

Conclusion

Human skin fibroblasts can internalize exosomes from human adipose-derived stem cells to promote their proliferation and migration.GelMA hydrogel with a concentration of 5% and loaded with exosomes from human adipose-derived stem cells （1×10⁸/ml） can release exosomes slowly and continuously to promote the proliferation of human skin fibroblasts， and it can be used as a good bio-carrier for exosomes.