诱导多能干细胞分泌的外泌体对角质细胞增殖与迁移的影响

Effects of exosomes secreted by induced pluripotent stem cells on keratinocyte proliferation and migration

目的探讨多能干细胞(iPSC)分泌的外泌体(Exos)促进角质细胞的增殖、侵袭与迁移,从而促进伤口愈合的作用。方法提取iPSC-Exos并通过透射电子显微镜、纳米颗粒追踪分析技术和蛋白质印迹法进行鉴定。将带有PKH26标记纯化的iPSC-Exos加到角质细胞(HaCaT)里进行角质细胞摄取外泌体的测定;用细胞计数试剂盒(CCK-8)检测外泌体最佳工作浓度。HaCaT细胞分为对照组(细胞划痕)和实验组(细胞划痕之后加入最佳工作浓度的外泌体)。分别用CCK-8、5-乙炔基-2′脱氧尿嘧啶核苷(EdU)、划痕实验及Transwell实验检测各组细胞的增殖、迁移以及侵袭能力。结果iPSC-Exos呈现圆形或椭圆形的膜囊泡结构,直径为(120.00±25.00)nm。实验组iPSC-Exos特征性表面标志物CD9、CD63、CD81的表达均呈阳性,对照组均呈阴性。HaCaT细胞能够摄取iPSC-Exos。干预24 h后,对照组和实验组的划痕愈合率分别为(25.70±1.07)%和(71.60±12.76)%,Transwell侵袭细胞数分别为(86.33±10.79)和(166.33±24.13)个,EdU阳性比例分别为(45.30±3.17)%和(78.10±6.29)%,实验组的上述指标与对照组比较,在统计学上差异均有统计学意义(均P<0.05)。结论多能干细胞分泌的外泌体能够促进角质细胞的增殖、迁移及侵袭,从而间接促进伤口愈合。

Objective To explore the role of exosomes secreted by induced pluripotent stem cells(iPSC)in promoting the proliferation,invasion and migration of keratinocytes,thereby facilitating wound healing.Methods Extract iPSC-Exos and identify them through transmissionelectron microscopy,nanoparticletracking analysis technology,and Western blotting.Purified iPSC-Exos labeled with PKH26 were added to keratinocytes(HaCaT)for the determination of keratinocyte uptake of exosomes.The optimal working concentration of exosomes was assessed using cell counting kit-8(CCK-8),and cells were divided into control group(cell scratch),and experimental group(cell scratch followed by addition of exosomes at the optimal working concentration).Proliferation,migration,and invasion abilities of cells in each group were evaluated using CCK-8,5-ethynyl-2′-deoxyuridine(EdU),scratch assay,and Transwell assay.Results iPSC-Exos exhibit a membranous vesicular structure with a round or elliptical shape,and their diameter is(120.00±25.00)nm.The expression of characteristic surface markers CD9,CD63,and CD81 on iPSC-Exos is positive in the experimental group,while being negative in the control group.HaCaT cells are capable of internalizing iPSC-Exos.After 24 hours of intervention,the scratch healing rates in the control and experimental groups are(25.70±1.07)%and(71.60±12.76)%,respectively.The Transwell invasion cell numbers are(86.33±10.79)and(166.33±24.13)in the control and experimental groups,and the EdU-positive proportions are(45.30±3.17)%and(78.10±6.29)%,respectively.The above indicators in the experimental group show statistically significant differences compared to the control group(all P<0.05).Conclusion The exosomes secreted by pluripotent stem cells can promote the proliferation,migration,and invasion of keratinocytes,thereby indirectly promoting wound healing.