芦可替尼通过骨髓源性外泌体抑制原发性骨髓纤维化的血管新生  

Ruxolitinib inhibits angiogenesis in primary myelofibrosis through bone marrow-derived exosomes

作　　者：梁嘉怡 李琼琼 陆丽娜 成志勇[2] 王素云 LIANG Jiayi;LI Qiongqiong;LU Lina;CHENG Zhiyong;WANG Suyun(Department of Hematology,Shenzhen Longhua District Central Hospital,Shenzhen 518000,Guangdong,China;Department of Hematology,Baoding No.1 Hospital,Baoding 071000,Hebei,China)

机构地区：[1]深圳市龙华区中心医院血液内科,深圳518000 [2]保定市第一医院血液内科,保定071000

出　　处：《海军军医大学学报》2024年第4期454-461,共8页Academic Journal of Naval Medical University

基　　金：深圳市科技计划:基础研究专项(自然科学基金)(JCYJ20210324141007017,JCYJ20230807151307015)。

摘　　要：目的探究芦可替尼能否在外泌体层面通过抑制血管新生减缓原发性骨髓纤维化(PMF)发展。方法采集3例初诊PMF患者治疗前(初诊组)和芦可替尼治疗3个月后(芦可替尼组)及3例原发免疫性血小板减少症患者(对照组)的骨髓液,提取外泌体。采用蛋白质印迹法检测外泌体中促血管新生相关因子的蛋白质表达水平。将外泌体与人脐静脉内皮细胞(HUVEC)共培养24 h后,采用CCK-8法检测HUVEC的增殖活力,采用流式细胞术检测HUVEC细胞周期分布,采用划痕实验和Transwell侵袭实验检测HUVEC的迁移和侵袭能力,采用qPCR和蛋白质印迹法检测HUVEC中侵袭增殖相关因子的mRNA和蛋白质表达水平。结果与对照组相比,初诊组外泌体中血管内皮生长因子(VEGF)、VEGF受体1(VEGFR1)、低氧诱导因子-1α(HIF-1α)、环氧合酶-2(COX-2)表达水平均增加(均P<0.01);芦可替尼组VEGF、VEGFR1、HIF-1α、COX-2表达水平均低于初诊组(均P<0.01)。与初诊组外泌体共培养的HUVEC增殖、迁移和侵袭能力相较于对照组均增强(均P<0.05),而与芦可替尼组外泌体共培养的HUVEC增殖、迁移和侵袭能力较初诊组下降(均P<0.05)。与初诊组外泌体共培养的HUVEC中S期细胞占比高于对照组,与芦可替尼组外泌体共培养的HUVEC中S期细胞占比低于初诊组。与对照组相比,与初诊组外泌体共培养的HUVEC中基质金属蛋白酶(MMP)-2、MMP-9、局部黏着斑激酶(FAK)、增殖细胞核抗原(PCNA)mRNA和蛋白质表达水平均较高(均P<0.01);与芦可替尼组外泌体共培养的HUVEC中MMP-2、MMP-9、FAK、PCNA mRNA和蛋白质表达水平均较初诊组下降(均P<0.01)。结论体外实验中,PMF初诊患者骨髓源性外泌体可通过血管新生机制促进PMF进展,芦可替尼能够在外泌体层面抑制血管新生从而发挥PMF治疗作用。Objective To investigate whether ruxolitinib can slow down the progression of primary myelofibrosis(PMF)by inhibiting angiogenesis at exosome level.Methods Exosomes were extracted from bone marrow samples collected from 3 newly diagnosed PMF patients before treatment(newly diagnosed group)and after 3 month of treatment with ruxolitinib(ruxolitinib group)and 3 primary immune thrombocytopenia patients(control group).The expression of angiogenesis-related factors in exosomes was detected by Western blotting.The exosomes were co-cultured with human umbilical vein endothelial cells(HUVECs)for 24 h.The proliferation ability of HUVECs was detected by cell counting kit 8 method.The cell cycle distribution of HUVECs was detected by flow cytometry.The migration and invasion of HUVECs were detected by scratch assay and Transwell invasion assay.The mRNA and protein expression levels of invasion-and proliferation-related factors in HUVECs were detected by quantitative polymerase chain reaction and Western blotting.Results Compared with the control group,the expression levels of vascular endothelial growth factor(VEGF),VEGF receptor 1(VEGFR1),hypoxia inducible factor-1α(HIF-1α)and cyclooxygenase-2(COX-2)in the exosomes were significantly higher in the newly diagnosed group(all P<0.01),while they were significantly lower in the ruxolitinib group than those in the newly diagnosed group(all P<0.01).Compared with the control group,the proliferation,migration and invasion of the HUVECs co-cultured with exosomes were significantly enhanced in the newly diagnosed group(all P<0.05),while they were significantly weakened in the ruxolitinib group than those in the newly diagnosed group(all P<0.05).The proportion of HUVECs co-cultured with exosomes at S phase was higher in the newly diagnosed group than that in the control group,while the proportion of HUVECs co-cultured with exosomes at S phase was lower in the ruxolitinib group than that in the newly diagnosed group.Compared with the control group,the mRNA and protein expression leve

关 键 词：原发性骨髓纤维化 外泌体 血管新生 芦可替尼 

分 类 号：R551.3[医药卫生—血液循环系统疾病]