脂肪间充质干细胞外囊泡通过增加LC3B表达对人微血管内皮细胞损伤的影响  

Effect of Extracellular Vesicles in Adipose-derived Mesenchymal Stem Cells on Human Microvascular Endothelial Cells Injury by Increasing LC3B Expression

作　　者：刘亮[1] 林煜[1] 林通[1] 杨建明 翁羽蕙 LIU Liang;LIN Yu;LIN Tong;YANG Jianming;WENG Yuhui(Fuqing City Hospital Affiliated to Fujian Medical University,Fuqing 350300,Fujian,China)

机构地区：[1]福建医科大学附属福清市医院,福建福清350300

出　　处：《中西医结合心脑血管病杂志》2024年第14期2559-2567,共9页Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease

摘　　要：目的:探讨低氧微环境诱导下脂肪间充质干细胞(ADSCs)外囊泡(Evs)通过增加微管相关蛋白轻链3B(LC3B)表达对人微血管内皮细胞损伤的保护作用。方法:分离大鼠ADSCs,并通过成骨、成脂分化和细胞表面标志物的表达进行鉴定。利用常氧(21%O2)和低氧(1%O2)条件分别获得常氧Evs和低氧Evs,通过形态和表面标志物的表达进行鉴定。构建心肌微血管内皮细胞(CMECs)细胞氧糖剥夺/再灌注(OGD/R)模型,并将CMECs分为对照组(Control组)、氧糖剥夺/再灌注组(OGD/R组)、常氧Evs组(N-Evs组,给予常氧Evs培养24 h)、低氧Evs组(H-Evs组,给予低氧Evs培养24 h)、低氧Evs+自噬抑制剂组(H-Evs+3-MA组,给予低氧Evs和3-MA 5 mmol/L培养24 h)。采用细胞计数试剂盒(CCK-8)法、5-乙炔基-2-脱氧尿嘧啶核苷(EdU)染色、划痕实验、Transwell实验、管腔形成实验和透射电镜分别检测CMECs增殖、迁移、侵袭、血管形成和自噬能力;采用酶联免疫吸附法(Western Blot)检测增殖细胞核抗原(PCNA)、Ki67、基质金属蛋白酶(MMP)-2/MMP-9、缺氧诱导因子-1α(HIF-1α)、血管内皮生长因子(VEGF)、LC3B、重组人自噬效应蛋白1(Beclin1)蛋白表达。结果:与Control组比较,OGD/R组CMECs增殖、迁移、侵袭、血管形成、自噬能力减弱(P<0.05),PCNA、Ki67、MMP-2/MMP-9、HIF-1α、VEGF、LC3B-Ⅱ/Ⅰ、Beclin1表达减少(P<0.05)。与OGD/R组比较,N-Evs组和H-Evs组CMECs增殖、迁移、侵袭、血管形成、自噬能力增强(P<0.05),PCNA、Ki67、MMP-2/MMP-9、HIF-1α、VEGF、LC3B-Ⅱ/Ⅰ、Beclin1表达增加(P<0.05),且H-Evs组优于N-Evs组(P<0.05)。与H-Evs组比较,H-Evs+3-MA组CMECs增殖、迁移、侵袭、血管形成、自噬能力减弱(P<0.05),PCNA、Ki67、MMP-2/MMP-9、HIF-1α、VEGF、LC3B-Ⅱ/Ⅰ、Beclin1表达减少(P<0.05)。结论:低氧微环境诱导下的ADSCs-Evs通过增加LC3B表达可促进OGD/R损伤CMECs的增殖、迁移、侵袭、血管形成和自噬能力。Objective:To investigate the protective effect of extracellular vesicles(Evs)in adipose-derived mesenchymal stem cells(ADSCs)induced by hypoxic microenvironment on human microvascular endothelial cells injury by increasing the expression of microtubule-associated protein light chain 3B(LC3B).Methods:The rat ADSCs were isolated and identified by osteogenic differentiation,lipogenic differentiation and expression of cell surface markers.Normoxic Evs and hypoxic Evs were obtained under normal oxygen(21%O 2)and hypoxic oxygen(1%O 2)conditions respectively,and identified by the expression of morphology and surface markers.The oxygen-glucose deprivation/reperfusion(OGD/R)model of cardiac microvascular endothelial cells(CMECs)was constructed,and CMECs were divided into Control group,OGD/R group,N-Evs group,hypoxic Evs group(H-EVS group,hypoxic Evs culture for 24 h),hypoxic Evs+autophagy inhibitor group(H-EVS+3-MA group,hypoxic Evs and 3-MA 5 mmol/L culture for 24 h).The proliferation,migration,invasion,angiogenesis and autophagy of CMECs were detected by cell counting kit(CCK-8),5-acetylidene-2-deoxyuracil nucleoside(EdU)staining,scratch test,Transwell test,tubular formation test and transmission electron microscopy.The expression of proliferating cell nuclear antigen(PCNA),Ki67,matrix metalloproteinase(MMP)-2/MMP-9,hypoxia-inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),LC3B and recombinant human autophagy effect protein 1(Beclin1)were detected by enzyme-linked immunosorbent assay.Results:Compared with the Control group,the proliferation,migration,invasion,angiogenesis and autophagy of CMECs in the OGD/R group were reduced(P<0.05),and the expressions of PCNA,Ki67,MMP-2/MMP-9,HIF-1α,VEGF,LC3B-Ⅱ/Ⅰ and Beclin1 decreased(P<0.05).Compared with the OGD/R group,the proliferation,migration,invasion,angiogenesis and autophagy of CMECs in the N-Evs group and H-Evs group were enhanced(P<0.05),and the expressions of PCNA,Ki67,MMP-2/MMP-9,HIF-1α,VEGF,LC3B-Ⅱ/Ⅰ and Beclin1 increased(P<0.05).These inde

关 键 词：心肌微血管内皮细胞 脂肪间充质干细胞 外囊泡 低氧微环境 大鼠 实验研究 

分 类 号：R542.2[医药卫生—心血管疾病]