ATG5介导内皮细胞外泌体调控平滑肌细胞增殖

ATG5 regulates VSMC proliferation by mediating endothelial exosomes

目的探究ATG5介导人脐静脉内皮细胞外泌体对小鼠胸主动脉平滑肌细胞增殖的影响。方法利用0.1%Ⅱ型胶原酶灌注消化法获得原代人脐静脉内皮细胞;利用组织块贴壁法分离原代小鼠胸主动脉平滑肌细胞;利用超速离心法提取内皮细胞外泌体;利用共聚焦高内涵成像分析仪示踪平滑肌细胞摄取内皮细胞外泌体;利用siRNA干扰内皮细胞ATG5蛋白表达;利用高内涵DPC成像模式分析平滑肌细胞增殖能力。结果成功得到活性好、纯度高的人脐静脉内皮细胞和小鼠胸主动脉平滑肌细胞。超速离心法获得的内皮细胞外泌体经免疫印迹鉴定,呈Hsp70、TSG101、CD9和CD63阳性。高内涵示踪结果显示平滑肌细胞在共培养30 min后开始摄取内皮细胞外泌体,共培养4 h后,平滑肌细胞摄取的外泌体明显增多。高内涵监测结果显示,与对照组比较,ATG5干扰组内皮细胞分泌的外泌体在浓度为200μg/ml时能够明显抑制平滑肌细胞的增殖。结论ATG5可通过介导内皮细胞外泌体调控平滑肌细胞增殖。

Objective To investigate ATG5 regulates VSMC proliferation by mediating endothelial exosomes.Methods HUVECs were separated by type II collagenase digestion at 37℃and then cultivated in DMEM/F-12 culture containing 10%FBS.Tissue explants adherent method was applied to obtain VSMCs.VSMCs was cultivated in DMEM/F-12 culture containing 20%FBS.The uptake of endothelial exosomes by VSMCs was tracked by high-content cell imaging system.SiRNA was used to knock down the expression of ATG5.Results HUVECs with high purity and good viability were isolated successfully by 0.1%typeⅡcollagenase digestion.The cell displayed typical cobblestone-like morphology under the inverted phase-contrast microscope.Immunofluorescence technique validated that the isolated cells with CD31/Pan-Cadherin/FactorⅧ-positive phenotype account for 96%.VSMCs were observed on the 3 rd day of the culture.The cells showed long shuttle and polygonal morphology.Immunofluorescence technique identified that the expression frequency of SM22-αandα-actin was about 95%.Endothelial exosomes obtained by ultracentrifugation displayed Hsp70/TSG101/CD9/CD63 positive phenotype.VSMCs started to uptake endothelial exosomes after 30 minutes co-culture showed by high-content cell imaging system.After 4 hours,the numbers of endothelial exosomes uptake by VSMCs increased significantly.Cell growth and proliferation curve showed that,at the concentration of 200μg/ml,the exosomes secreted by HUVEC in which ATG5 decreased could inhibit the proliferation of VSMC.Conclusion ATG5 regulates VSMC proliferation by mediating endothelial exosomes.