脐静脉内皮细胞外泌体对炎症因子刺激下前软骨细胞凋亡的影响

doi:10.3969/j.issn.1674-8115.2021.02.004

摘要/Abstract

摘要：

目的·探讨脐静脉内皮细胞外泌体对炎症刺激下小鼠前软骨细胞ATDC5凋亡的影响。方法·采用试剂盒分离人脐静脉内皮细胞（human umbilical vein endothelial cell，HUVEC）外泌体，采用Western blotting检测外泌体标志蛋白肿瘤易感基因101（tumor susceptibility gene 101，Tsg101）、白细胞分化抗原9（cluster differentiation 9，CD9）、凋亡相关基因2互作蛋白X（apoptosis linked gene-2-interacting protein X，Alix）的表达水平。透射电镜观察外泌体形态，粒度检测鉴定外泌体大小。使用荧光显微镜观察外泌体被小鼠前软骨细胞ATDC5摄取过程和活性氧（reactive oxygen species，ROS）的生成情况。TUNEL染色和流式细胞术检测外泌体对白细胞介素-1β（interleukin-1β，IL-1β）刺激下的ATDC5细胞凋亡的影响。Western blotting检测外泌体对于ATDC5细胞中凋亡相关蛋白B淋巴细胞瘤2（B-cell lymphoma/leukemia-2，Bcl-2）、Bcl-2相关X蛋白（Bcl-2 associated X protein，Bax）、裂解的半胱天冬酶3（cleaved caspase-3，c-caspase-3）和抗氧化应激相关蛋白核因子E2相关因子2（nuclear factor E2 related factor 2，Nrf-2）、Kelch样环氧氯丙烷相关蛋白1（Kelch-like ECH-associated protein 1，Keap-1）、血红素加氧酶1（heme oxygenase 1，HO-1）、还原型烟酰胺腺嘌呤二核苷酸醌氧化还原酶1（NADPH quinone oxidoreductase-like protein 1，NQO-1）表达水平的影响。结果·透射电镜下观察到HUVEC来源的外泌体呈椭圆形，中空，双层膜，并且阳性表达外泌体标志物CD9、Alix、Tsg101。与IL-1β刺激下的ATDC5细胞相比，外泌体促进炎症因子作用下的ATDC5细胞内ROS的生成（P=0.000）和凋亡的发生（P=0.000），Bax、c-caspase-3、Keap-1表达升高，Bcl-2、Nrf-2、HO-1、NQO-1表达降低。结论·HUVEC来源的外泌体可能通过抑制ATDC5细胞抗氧化应激的能力，促进IL-1β刺激下ATDC5细胞凋亡的发生。

关键词: 外泌体, 骨关节炎, 前软骨细胞, 脐静脉内皮细胞, 氧化应激, 凋亡

Abstract:

Objective·To investigate the effect of exosomes derived from umbilical vein endothelial cells (HUVECs) on apoptosis of murine pre-chondrogenic cell line ATDC5 cells under inflammatory stimulation.

Methods·The exosomes derived from HUVECs were isolated by using an exosome isolation kit. Western blotting was used to detect the exosome marker proteins, including tumor susceptibility gene 101 (Tsg101), cluster differentiation 9 (CD9) and apoptosis linked gene-2-interacting protein X (Alix). The morphology of exosomes was observed by transmission electron microscope, and the size of exosomes was identified by particle size detection. Fluorescence microscope was used to observe the ATDC5 cell uptake of exosomes and the production of reactive oxygen species (ROS). TUNEL staining and flow cytometry were used to examine the effect of exosomes on ATDC5 cell apoptosis stimulated by interleukin-1β (IL-1β). Western blotting was used to detect the effect of exosomes on the expression levels of ATDC5 apoptosis-related proteins such as B-cell lymphoma/leukemia 2 (Bcl-2), Bcl-2 associated X protein (Bax), cleaved caspase-3 (c-caspase-3) and anti-oxidative stress-related proteins such as nuclear factor E2 related factor 2 (Nrf-2), Kelch-like ECH-associated protein 1 (Keap-1), heme oxygenase 1 (HO-1) and NADPH quinone oxidoreductase-like protein 1 (NQO-1) under IL-1β stimulation.

Results·Under the transmission electron microscope, the HUVEC-derived exosomes were oval, hollow, double-layered, and positively expressed exosome markers CD9, Alix and Tsg101. Compared with the ATDC5 cells stimulated by IL-1β, ATDC5 cells stimulated by IL-1β incubated with exosomes had higher level of ROS (P=0.000) and higher apoptosis rate (P=0.000). The expression of Bax, c-caspase-3 and Keap-1 increased, and the expression of Bcl-2, Nrf-2, HO-1 and NQO-1 decreased in ATDC5 cells exposed to IL-1β and exosomes compared to ATDC5 cells only exposed to IL-1β.

Conclusion·HUVEC-derived exosomes may promote ATDC5 cells apoptosis under the stimulation of IL-1β by inhibiting the ability of ATDC5 cell to resist oxidative stress.

Key words: exosome, osteoarthritis (OA), pre-chondrogenic cell, human umbilical vein endothelial cell (HUVEC), oxidative stress, apoptosis

中图分类号:

R684.3

杨润泽, 许文宁, 郑火亮, 蒋盛旦. 脐静脉内皮细胞外泌体对炎症因子刺激下前软骨细胞凋亡的影响[J]. 上海交通大学学报(医学版), 2021, 41(2): 147-153.

Run-ze YANG, Wen-ning XU, Huo-liang ZHENG, Sheng-dan JIANG. Effects of exosomes derived from human umbilical vein endothelial cells on apoptosis of pre-chondrogenic cells stimulated by inflammatory factors[J]. JOURNAL OF SHANGHAI JIAOTONG UNIVERSITY (MEDICAL SCIENCE), 2021, 41(2): 147-153.

图/表 6

图1 HUVEC来源外泌体的鉴定Note:A. HUVEC-derived exosomes were observed by transmission electron microscopy. B. The expression of the exosome markers TSG101, CD9 and Alix was confirmed by Western blotting. C. The size distribution of HUVEC-derived exosomes.

Fig 1 Identification of HUVEC-derived exosomes

图2 荧光染料PKH26标记的外泌体与ATDC5细胞共培养3 h和6 h后的摄取情况

Fig 2 Uptaking of PKH26-labeled exosomes co-cultured with ATDC5 cells for 3 h and 6 h

图3 ATDC5细胞内ROS检测Note: A. Detection of ROS production by DCFH-DA assay. B. Quantitative analysis of the fluorescence intensity of ATDC5 cells under different treatment conditions. exo—exosomes. ①P=0.000.

Fig 3 Detection of ROS in ATDC5 cells

图4 TUNEL法检测ATDC5细胞凋亡Note:A. TUNEL staining of ATDC5 cells. B. Comparison of cell apoptosis rate among NC group, IL-1β group and IL-1β+exo group. exo—exosomes. ①P=0.000.

Fig 4 Detection of apoptosis of ATDC5 cells by TUNEL assay

图5 流式细胞术检测ATDC5细胞凋亡Note:A. Analysis of cell apoptosis by Annexin V-FITC/PI staining and flow cytometry analysis. B. Quantitative analysis of total cell apoptosis rate in NC group, IL-1β group and IL-1β+exo group. exo—exosomes. ①P=0.000.

Fig 5 Detection of apoptosis of ATDC5 cells by flow cytometry

图6 Western blotting检测ATDC5细胞中凋亡及抗氧化应激相关蛋白的表达Note:A. The expression of Bax, Bcl-2 and c-caspase-3 verified by Western blotting. B. The expression of Keap-1, Nrf-2, NQO-1 and HO-1 verified by Western blotting. C. Quantitative analysis of apoptosis-related protein expression changes in response to IL-1β stimulation and after incubation of ATDC5 cells with exsomes. D. Quantitative analysis of anti-oxidative stress-related protein expression changes in response to IL-1β stimulation and after incubation of ATDC5 cells with exosomes. ①P=0.009, ②P=0.043, ③P=0.005, ④P=0.037, ⑤P=0.003, ⑥P=0.038, ⑦P=0.002, ⑧P=0.001, ⑨P=0.009, ⑩P=0.039, ?P=0.001, ?P=0.033 ,?P=0.017, ?P=0.000.

Fig 6 Detection of the expression of apoptosis- and anti-oxidative stress-related proteins in ATDC5 cells by Western blotting

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