缺氧H9c2来源外泌体对HUVEC增殖、迁移和成管能力的影响

doi:10.11958/20231930

天津医药 ›› 2024, Vol. 52 ›› Issue (7): 714-719.doi: 10.11958/20231930

缺氧H9c2来源外泌体对HUVEC增殖、迁移和成管能力的影响

杨睿(), 魏琼, 孙逸坤, 赵梦竹, 程序, 刘梦华, 张冬梅^∆()

北京中医药大学东直门医院/中医内科学教育部和北京市重点实验室（邮编100700）

收稿日期:2024-01-10 修回日期:2024-03-05 出版日期:2024-07-15 发布日期:2024-07-11
通讯作者: ^∆E-mail：chaweto@126.com
作者简介:杨睿（1993），女，主治中医师，主要从事中医药防治心血管疾病方面研究。E-mail：Reyoung12@126.com
基金资助:
国家自然科学基金资助项目(81973780)

Effects of hypoxia H9c2 exosome on proliferation，migration and tube formation of HUVEC

YANG Rui(), WEI Qiong, SUN Yikun, ZHAO Mengzhu, CHENG Xu, LIU Menghua, ZHANG Dongmei^∆()

Dongzhimen Hospital, Ministry of Education and Beijing Key Laboratory of Traditional Chinese Medicine Internal Medicine, Beijing University of Chinese Medicine, Beijing 100700, China

Received:2024-01-10 Revised:2024-03-05 Published:2024-07-15 Online:2024-07-11
Contact: ^∆E-mail：chaweto@126.com

杨睿, 魏琼, 孙逸坤, 赵梦竹, 程序, 刘梦华, 张冬梅. 缺氧H9c2来源外泌体对HUVEC增殖、迁移和成管能力的影响[J]. 天津医药, 2024, 52(7): 714-719.

YANG Rui, WEI Qiong, SUN Yikun, ZHAO Mengzhu, CHENG Xu, LIU Menghua, ZHANG Dongmei. Effects of hypoxia H9c2 exosome on proliferation，migration and tube formation of HUVEC[J]. Tianjin Medical Journal, 2024, 52(7): 714-719.

摘要/Abstract

摘要：

目的探讨缺氧状态下大鼠心肌细胞H9c2来源外泌体对血管新生的影响。方法采用混合气体法制备H9c2细胞缺氧模型，以正常培养细胞为对照。分别提取2组细胞分泌的外泌体，采用纳米颗粒跟踪分析检测外泌体的浓度和粒径，透射电子显微镜观察外泌体的形态和大小，Western blot验证外泌体标志蛋白。制备人脐静脉内皮细胞（HUVEC）缺氧模型，并与H9c2外泌体共同孵育，分为常氧组、缺氧组、缺氧+正常H9c2外泌体（EXO-C）组和缺氧+缺氧H9c2外泌体（EXO-M）组。通过CCK-8法、细胞划痕实验、Matrigel体外三维成形实验分别检测HUVEC增殖、迁移、成管能力。结果外泌体鉴定结果显示常氧组和缺氧组H9c2外泌体样本颗粒浓度均处于1×10⁷~1×10¹²/mL，粒径大小40~160 nm；形态特征均为球形或茶托状结构，大小均一，形态完整；外泌体标志蛋白肿瘤易感基因101（TSG101）、CD63、CD9均有表达，无阴性蛋白钙联蛋白（Calnexin）的表达。与常氧组比，缺氧组HUVEC增殖能力、迁移面积、迁移率均显著下降，参与成管的长度及分支数、节点数均减少（P<0.01）；与缺氧组比，EXO-M组HUVEC细胞增殖能力下降，迁移面积减少、迁移率下降，参与成管长度及分支数进一步减少（P<0.05）；与EXO-C组相比，EXO-M组增殖能力下降，细胞迁移面积减少，迁移率下降（P<0.01）。结论缺氧H9c2来源的外泌体对HUVEC增殖、迁移、成管能力有一定程度的抑制作用。

关键词: 外泌体, 心肌梗死, 内皮细胞, 低氧, 血管新生

Abstract:

Objective To investigate the role of H9c2-derived exosomes in regulating angiogenesis in rat cardiomyocytes under hypoxia. Methods The hypoxia model of H9c2 cells was prepared by mixed gas method (the hypoxia model group), and the normal cultured cells were used as the control group. The exosomes secreted by the two groups of cells were extracted respectively. The concentration and particle size of exosomes were detected by nanoparticle tracking analysis. The morphology and size of exosomes were detected by transmission electron microscopy. Western blot assay was used to verify the exosome marker proteins. The hypoxia model of human umbilical vein endothelial cells (HUVEC) was established. HUVECs were incubated with H9c2 exosomes and divided into the normoxia group, the hypoxia group, the hypoxia + normal H9c2 exosomes (EXO-C) group and the hypoxia + hypoxia H9c2 exosomes (EXO-M) group. The proliferation, migration and tube formation of HUVECs were detected by CCK-8 method, cell scratch test and Matrigel in vitro three-dimensional forming test. Results The results of exosome identification showed that the particle concentration of H9c2 exosome samples was 1×10⁷-1×10¹²particles/mL and the particle size was 40-160 nm in the normoxia group and the hypoxia group. The morphological characteristics were spherical or saucer-like structure, uniform in size and complete in shape. Exosome marker proteins TSG101, CD63 and CD9 were expressed, and there was no expression of negative protein Calnexin. Compared with the normoxic group, the proliferation ability, migration area and migration rate of HUVEC were significantly decreased in the hypoxic group, and the length of tube, the number of branches and the number of nodes were decreased (P<0.01). Compared with the hypoxia group, the proliferation ability of HUVEC cells was decreased, the migration area was decreased, the migration rate was decreased and the length and number of branches involved in tube formation were further decreased in the EXO-M group (P<0.05). Compared with the EXO-C group, the proliferation ability of the EXO-M group decreased, the cell migration area decreased and the migration rate decreased (P<0.01). Conclusion Exosomes derived from hypoxic H9c2 can inhibit the proliferation, migration and tube formation of HUVEC.

Key words: exosomes, myocardial infarction, endothelial cells, hypoxia, angiogenesis

中图分类号:

R54，R363

图/表 10

图1 不同缺氧时间对H9c2细胞存活率的影响 a与缺氧0 h比较，b与缺氧24 h比较，c与缺氧30 h比较，P<0.05。

Fig.1 Effects of different hypoxia time on the survival rate of H9c2 cells

图2 H9c2外泌体的NTA鉴定

Fig.2 NTA identification of H9c2 exosomes

图3 H9c2外泌体的透射电镜鉴定（×30 000）

Fig.3 Identification of H9c2 exosomes by transmission electron microscopy (×30 000)

图4 H9c2外泌体标志蛋白的鉴定 A：常氧H9c2外泌体；B：缺氧H9c2外泌体；C：常氧H9c2细胞；D：缺氧H9c2细胞。

Fig.4 Identification of H9c2 exosome marker protein

图5 不同浓度H9c2外泌体对HUVEC细胞活力的影响 a与对照组（0 mg/L EXO-C组）比较，b与对照组（0 mg/L EXO-M组）比较，c与EXO-C比较，P<0.05。

Fig.5 Effects of different concentrations of H9c2 exosomes on HUVEC cell viability

图6 各组HUVEC细胞增殖能力的比较 a与常氧组比较，b与缺氧组比较，c与EXO-C组比较，P<0.05。

Fig. 6 Comparison of proliferation ability of HUVEC cells between four groups

表1 各组HUVEC细胞迁移能力的比较（n=6，$\bar{x}±s$）

Tab.1 Comparison of migration ability of HUVEC cells between four groups

组别	迁移面积/mm²	迁移率/%
常氧组	38.93±4.82	77.72±7.56
缺氧组	28.56±5.32^a	54.40±9.19^a
EXO-C组	25.12±2.54^a	48.05±6.11^a
EXO-M组	19.41±3.19^abc	37.23±7.54^abc
F	23.673^＊＊	29.821^＊＊

图7 各组HUVEC细胞迁移能力的比较（×40）

Fig. 7 Comparison of migration ability of HUVEC cells between four groups (×40)

图8 各组HUVEC细胞成管能力的比较（×100）

Fig. 8 Comparison of tube formation ability of HUVEC cells between four groups (×100)

表2 各组HUVEC细胞成管能力的比较（n=6，$\bar{x}±s$）

Tab. 2 Comparison of tube formation ability of HUVEC cells between four groups

组别	分支长度/cm	分支个数/（×10²个）	节点/（×10³个）
常氧组	27.66±9.88	5.22±1.67	3.40±1.41
缺氧组	14.20±3.22^a	3.13±0.58^a	1.46±0.31^a
EXO-C组	8.48±1.71^a	2.14±0.43^a	0.89±0.22^a
EXO-M组	7.10±0.88^ab	2.00±0.35^ab	0.73±0.08^a
F	18.910^＊＊	15.498^＊＊	17.061^＊＊

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缺氧H9c2来源外泌体对HUVEC增殖、迁移和成管能力的影响

Effects of hypoxia H9c2 exosome on proliferation，migration and tube formation of HUVEC

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