牙髓干细胞来源胞外囊泡通过调节miR-100-5p抑制γ射线所致小鼠骨髓细胞FDC-P1凋亡  

Dental pulp stem cells derived extracellular vesicles inhibitγ-ray induced apoptosis of mouse bone marrow cells FDC-P1 by regulating miR-100-5p

作　　者：易静 陶宁 吕琳 刘超 张愉宁 段晗 王华 YI Jing;TAO Ning;LYU Lin;LIU Chao;ZHANG Yu-ning;DUAN Han;WANG Hua(College of Life Sciences,Anhui Medical University,Hefei 230032,China;Institute of Radiation Medicine,Academy of Military Medical Sciences,Beijing 100850,China;The First Affiliated Hospital of Jinan University,Guangzhou 510630,China)

机构地区：[1]安徽医科大学生命科学学院,安徽合肥230032 [2]军事科学院军事医学研究院辐射医学研究所,北京100850 [3]暨南大学附属第一医院,广东广州510630

出　　处：《中国药理学与毒理学杂志》2022年第5期355-363,共9页Chinese Journal of Pharmacology and Toxicology

摘　　要：目的探讨牙髓干细胞来源胞外囊泡(DPSC-EV)对γ射线导致小鼠骨髓细胞凋亡的抑制作用及机制。方法使用超速离心法从DPSC中分离DPSC-EV,用Western印迹法鉴定其表面标志物,用纳米颗粒跟踪分析技术(NTA)检测DPSC-EV颗粒数目及粒径大小。细胞处理:①^(60)Coγ射线照射小鼠骨髓细胞FDC-P1,单次照射剂量分别为0,2和6 Gy,实验时间为照射后24和48 h;②FDC-P1细胞经^(60)Coγ射线2和6 Gy照射后立即加入DPSC-EV(终浓度5×10^(11) L^(-1))干预24 h;③FDC-P1细胞转染微RNA(miRNA)抑制剂后进行2 Gy照射并立即加入DPSC-EV(终浓度5×10^(11) L^(-1))干预24 h;④FDC-P1细胞转染miRNA模拟物后进行2 Gy照射,继续培养24 h。用流式细胞术检测细胞凋亡率,Western印迹法检测胱天蛋白酶3、活化胱天蛋白酶3、聚腺苷二磷酸核糖聚合酶(PARP)和活化PARP蛋白表达水平,逆转录实时定量PCR检测细胞miR-100-5p表达水平。结果①与细胞对照组相比,^(60)Coγ射线2和6 Gy照射后24和48 h,FDC-P1细胞凋亡率均显著升高(P<0.01),细胞中胱天蛋白酶3和PARP蛋白活化水平均显著升高(P<0.05,P<0.01)。②照射后加DPSC-EV处理24 h,FDC-P1细胞凋亡率较2和6 Gy单纯照射组均明显降低(P<0.01),PARP蛋白活化水平显著降低(P<0.05,P<0.01)。③经γ射线2 Gy照射后24 h,FDC-P1细胞miR-100-5p表达水平与细胞对照组比较显著降低(P<0.01),DPSC-EV干预可逆转2 Gy照射导致的miR-100-5p表达水平降低(P<0.01);转染miR-100-5p抑制剂后,FDC-P1细胞经2 Gy照射并加入DPSC-EV后,与抑制剂对照组相比,细胞凋亡率显著升高(P<0.01);④转染miR-100-5p模拟物后,2 Gy照射,与模拟物对照组相比,FDC-P1细胞凋亡率明显降低(P<0.01)。结论DPSC-EV可能通过上调骨髓细胞中miR-100-5p表达水平而抑制γ射线所致小鼠骨髓细胞凋亡。OBJECTIVE To investigate the protective effect and mechanism of dental pulp stem cells(DPSCs)derived extracellular vesicles(DPSC-EVs)on irradiation-induced apoptosis of mouse bone marrow cells.METHODS DPSC-EVs were isolated from DPSCs by ultracentrifugation.Western blotting was used to identify the surface markers and nanoparticle tracking analysis(NTA)was used to detect the number and size of the particles.①Mouse bone marrow cells FDC-P1 were irradiated with ^(60)Coγray and divided into 24 and 48 h groups according to the duration of irradiation,or into 0,2 and 6 Gy groups according to the irradiation doses.②DPSC-EVs at the terminal concentration of 5×10^(11) L^(-1) was added immediately into FDC-P1 cells after 2 and 6 Gy irradiation and then cultured for 24 h.③FDC-P1 cells transfected with miR-100-5p inhibitor were irradiated with 2 Gy and immediately treated with DPSC-EVs at the terminal concentration of 5×10^(11) L^(-1) for 24 h.④FDC-P1 cells transfected with miR-100-5p mimic were irradiated with 2 Gy and cultured for 24 h after irradiation.The apoptosis rate of FDC-P1 cells was detected by flow cytometry and the protein expression levels of caspase 3,cleaved-caspase 3,poly ADP-ribose polymerase(PARP)and cleaved-PARP were detected by Western blotting.The expression level of miR-100-5p in FDC-P1 cells was detected by real time-quantitative polymerase chain reaction(RT-qPCR).RESULTS①Compared with the control group,the apoptosis rates of FDC-P1 cells were significantly increased in 2 and 6 Gy groups at 24 or 48 h after irradiation(P<0.01),and the active levels of caspase 3 and PARP proteins were significantly upregulated(P<0.05,P<0.01).②After irradiation and DPSC-EVs treatment for 24 h,the apoptosis rates of FDC-P1 cells were significantly reduced(P<0.01)and the active level of PARP protein was significantly down-regulated(P<0.05,P<0.01)compared with 2 and 6 Gy irradiation group.③At 24 h after irradiation,the expression level of miR-100-5p was significantly decreased in 2 Gy group(P<0.01),which

关 键 词：放射性损伤 细胞凋亡 间充质干细胞 胞外囊泡 

分 类 号：R979.6[医药卫生—药品] R963[医药卫生—药学]