缺氧心肌细胞来源外泌体对Gli1^+细胞纤维化表型转换的作用机制  

The fibrotic effect of hypoxic cardiomyocyte-derived exosomes on Gli1^+ cells and its mechanism

作　　者：林潮金 秦爱萍 李松沛 霍然 邓赛 傅小媚 向杨 余细勇 LIN Chao-jin;QIN Ai-ping;Li Song-pei;HUO Ran;DENG Sai;FU Xiao-mei;XIANG Yang;YU Xi-yong(School of Pharmaceutical Sciences of Guangzhou Medical University,Guangdong Key Lab of Molecular Target & ClinicalPharmacology,State Key Lab of Respiratory Diseases Pharmacology Group,Guangzhou 511436,China)

机构地区：[1]广州医科大学药学院广东省分子靶标与临床药理学重点实验室呼吸疾病国家重点实验室药理学组

出　　处：《中国药理学通报》2019年第11期1509-1515,共7页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金重点项目(No U1601227,81330007);广东省重大科技计划资助项目(No GD2015B020225006)

摘　　要：目的 观察缺氧心肌细胞来源外泌体对Gli1 +细胞纤维化过程的影响,并探讨其可能的机制。方法 分离Gli1 +细胞和SD乳鼠心肌细胞。心肌细胞分别进行常氧和缺氧培养,收集培养基并提取外泌体,用透射电镜、Nanosight、Western blot等方法对其鉴定。两种外泌体分别与Gli1 +细胞共培养。RT-qPCR法检测这两种外泌体并对比分析,明确其中起关键调控作用的miRNA。转染miRNA模拟物观察Gli1 +细胞中纤维化相关蛋白的表达水平。结果 Gli1 +细胞强阳性表达CD29、CD105和Gli1,不表达CD31、CD34和CD45。心肌细胞表达cTnT和α-Actinin,其外泌体直径约100 nm,并检测到Flotillin-1、Alix、HSP-70和CD63。缺氧处理外泌体中miR-223明显上调( P <0.01);Gli1 +细胞经缺氧外泌体和 miR-223 mimic处理,纤维化相关的蛋白α-SMA( P <0.01, P <0.05)、DDR-2( P <0.01, P <0.01)和collagen I( P <0.05, P <0.01)的表达都明显升高。结论 缺氧心肌细胞外泌体能促进Gli1 +细胞内纤维化相关蛋白的表达,使其向纤维化表型转换,这一作用可能与外泌体中高表达的miR-223有关。Aim To investigate whether the Gli1^+cells fibrosis is mediated by damaged cardiomyocyte-derived exosomes and explore the possible mechanism. Methods Gli1^+cells were isolated from mouse heart and identified by flow cytometry. Neonatal rat cardiomyocytes were subjected to normoxic and hypoxic treatment,respectively. Normoxic/hypoxic exosomes were obtained and detected by flow cytometry,nanosight tracficking analysis(NTA),transmission electron microscopy(TEM) and Western blot. Key exosomal miRNA content was determined by RT-qPCR. Then Gli1^+cells were co-cultured with normoxic/hypoxic exosomes or transfected with miRNA mimic to confirm the expression level of fibrosis-related proteins. Flow cytometry was used to detect the proportion of Gli1^+cells positively expressing both DDR-2 and ɑ-SMA protein. Results Gli1^+cells biomarkers,including CD29,CD105 and Gli1,were identified,but CD31,CD34 and CD45 were undetectable. NTA showed that the diameters of exosomes were about 100 nm and exosomal markers CD63,HSP-70, Alix and Flotillin-1 were detectable by Western blot. Further study found that cardiomyocytes produced more exosomal miR-223(P<0.01) under hypoxia treatment. Hypoxic exosomes and miR-223 mimic obviously increased the expression of α-SMA(P<0.01,P<0.05),DRR-2(P<0.01,P<0.01) and collagen I(P<0.05,P<0.01) in Gli1^+cells. Conclusions Hypoxic cardiomyocyte-derived exosomes may promote Gli1^+ cell fibrosis,which may be related to the high expression of miR-223 in exosomes.

关 键 词：心肌细胞 GLI1 +细胞 缺氧 外泌体 miRNA 纤维化 

分 类 号：R32[医药卫生—人体解剖和组织胚胎学] R322.11[医药卫生—基础医学]