低强度脉冲超声联合脂肪间充质干细胞源性外泌体促进内皮细胞血管生成的实验研究

Experimental study of low-intensity pulsed ultrasound combined with adipose mesenchymal stem cells derived exosomes to enhance the angiogenesis of endothelial cells

目的探讨低强度脉冲超声(LIPUS)联合脂肪间充质干细胞(ADSCs)源性外泌体(ADSCs-Exos)对内皮细胞血管生成能力的影响。方法取SD大鼠腹股沟处脂肪,采用酶消化法分离培养得到ADSCs,并对其表面分子进行鉴定。收集第3代ADSCs上清液提取ADSCs-Exos,应用透射电子显微镜、纳米颗粒跟踪分析仪、蛋白免疫印迹法观察其特征。将人脐静脉内皮细胞(HUVECs)按照不同处理方式分为对照组、外泌体组、LIPUS组及LIPUS+外泌体组,采用CCK-8实验、细胞划痕实验及小管形成实验检测不同处理方式对HUVECs增殖、迁移及成管能力的影响。结果ADSCs形态呈梭形,表面蛋白CD34、CD45为阴性,CD29、CD90为阳性。ADSCs-Exos为形态一致的茶托样囊泡,表达标志性蛋白CD9和Tsg101,其粒径97.8%集中在(118.8±80.8)nm。CCK-8实验显示LIPUS+外泌体组吸光度值为1.882±0.138,明显高于外泌体组、LIPUS组及对照组(0.646±0.034、0.631±0.027、0.462±0.036),差异均有统计学意义(均P<0.05)。细胞划痕实验显示LIPUS+外泌体组细胞迁移率为(87.7±4.5)%,均明显高于外泌体组、LIPUS组及对照组[(43.0±5.1)%、(34.7±2.3)%、(25.1±2.0)%],差异均有统计学意义(均P<0.05)。小管形成实验显示LIPUS+外泌体组HUVECs形成管腔数量及周长均大于外泌体组、LIPUS组及对照组,差异均有统计学意义(均P<0.05)。结论LIPUS联合ADSCs-Exos可有效促进血管内皮细胞的增殖、迁移及成管,具有促进血管生成能力的作用。

ObjectiveTo explore the effect of low-intensity pulsed ultrasound(LIPUS)combined with adipose mesenchymal stem cells(ADSCs)derived exosomes(ADSCs-Exos)on endothelial cell angiogenesis.MethodsAdipose tissue in the groin of SD rats was obtained.The ADSCs were obtained by enzymatic digestion and the surface molecules were identified.The ADSC-Exos were extracted from the supernatant of the 3 rd generation ADSCs,and its characteristic was observed by transmission electron microscopy,nanoparticle tracking analysis,and Western blotting.Human umbilical vein endothelial cells(HUVECs)were divided into control group,exosome group,LIPUS group and LIPUS+exosome group according to different treatment methods,the effects of different treatments on the proliferation,migration and tube formation ability of HUVECs were detected by cell counting kit 8(CCK-8)assay,scratch test and tubule formation test.ResultsThe shape of ADSCs was shuttlelike,the surface protein CD34 and CD45 were negative,CD29 and CD90 were positive.ADSC-Exos were saucer shaped vesicle with uniform morphology,and expressed the membrane-labeled proteins CD9 and Tsg101,with a particle size of 97.8%concentrated in(118.8±80.8)nm.CCK-8 assay showed that the optical density value of the LIPUS+exosome group was 1.882±0.138,which was significantly higher than that of the exosome group,LIPUS group and control group(0.646±0.034,0.631±0.027 and 0.462±0.036),and the differences were statistically significant(all P<0.05).The scratch test showed that the cell migration rate of the LIPUS+exosome group was(87.7±4.5)%,which was significantly higher than that of the exosome group,LIPUS group and control group[(43.0±5.1)%,(34.7±2.3)%and(25.1±2.0%)],the differences were statistically significant(all P<0.05).The tube formation test showed that the tube formation ability of HUVECs in the LIPUS+exosome group was higher than that of the exosomes group,LIPUS group and control group,and the differences were statistically significant(all P<0.05).ConclusionLIPUS combined with ADSCs-Exos can effectively promote the proliferation,migration and tube formation of vascular endothelial cells,and has the effect of enhancing angiogenesis.