不同存储温度的huMSC-exo对UVB辐照HaCaT细胞的修复作用研究

The recovery effects of huMSC-exo stored at different temperatures on UVB irradiated HaCaT cells

目的探究了不同存储温度的huMSC-exo对UVB辐照HaCaT细胞的修复作用,为外泌体存储选择适宜的温度条件提供理论依据。方法采用超速离心法从huMSC培养基上清中提取外泌体,用Western blot、纳米颗粒跟踪分析技术和透射电镜技术进行表征,并置于–80、–20、4、25和45℃存储1、3、7、14、30、50、100和150 d。构建UVB辐照HaCaT光老化细胞模型,用huMSC-exo处理24 h后观察细胞形态、检测细胞存活率、SA-β-gal活性、T-SOD活性、MDA和LDH含量。结果huMSC-exo呈具有清晰膜的茶托样结构,尺寸中位数为114 nm,30~150 nm囊泡数量占比约61.42%,表达外泌体表面标记蛋白CD9、CD63和CD81。–80℃和25℃存储1~150 d的huMSC-exo能显著提高UVB辐照HaCaT细胞存活率、恢复细胞形态、降低SA-β-gal活性、部分恢复细胞T-SOD、MDA和LDH水平;–20℃存储的huMSC-exo对HaCaT的增殖活性、细胞形态和SA-β-gal活性有恢复作用;4℃存储的huMSC-exo能减轻细胞老化水平;45℃存储的huMSC-exo无显著修复作用。结论–80℃、25℃存储的huMSC-exo能够显著恢复UVB辐照引起的HaCaT损伤,–20℃、4℃huMSC-exo作用效果次之,45℃的huMSC-exo无显著影响,提示–80℃和25℃能够较好地维持huMSC-exo的生物学功能。

Objective To investigate the recovery effects of huMSC-exo stored at different temperatures on UVB irradiated HaCaT cells.Methods Exosomes were extracted from the medium supernatant of huMSC by ultracentrifugation and characterized by Western blot,nanoparticle tracking analysis and transmission electron microscopy.huMSC-exo were separated and stored at–80℃,–20℃,4℃,25℃and 45℃for 1 day,3 days,7 days,14 days,30 days,50 days,100 days and 150 days.huMSC-exo was then added to UVB irradiated HaCaT cells.After 24 h incubation,cell viability of haCaT was measured by CCK8 assay and cell morphology was photographed.The activities of SA-β-gal and T-SOD,the contents of MDA and LDH were detected by commercial kit.Results The huMSC-exo showed a typical saucer-like structure with clear membrane.The median particle size was 114 nm,and the proportion of vesicles within the size range of 30-150 nm was about 61.42%.Besides,huMSC-exo expressed marker proteins CD9,CD63 and CD81.huMSC-exo stored at-80℃and 25℃for 1-150 d significantly improved the cell viability of UVB irradiated HaCaT cells,reduced the activity of SA-β-gal,and restored cell morphology,T-SOD,MDA and LDH levels.huMSC-exo stored at-20℃also showed beneficial effects on the cell proliferation,morphology and cell aging of HaCaT cells.huMSC-exo stored at 4℃decreased SA-β-gal activity.However,huMSC-exo stored at 45℃could not recover UVB induced damage of HaCaT cells.Conclusion huMSC-exo stored at-80℃and 25℃shows a significant repair effect on UVB irradiated HaCaT cells,followed by huMSC-exo stored at-20℃and 4℃,while huMSC-exo stored at 45℃exerts no significant recovery effect on HaCaT cells.These results suggests that the biological functions of huMSC-exo can be relatively well maintained at-80℃and 25℃.