| 引用本文: |
刘颖,尹园缘,邹巍莹,曹晖,宾东华.象皮生肌膏预处理的骨髓间充质干细胞源外泌体对人脐静脉内皮细胞血管生成的影响[J].湖南中医药大学学报,2023,43(2):249-256[点击复制] |
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| 象皮生肌膏预处理的骨髓间充质干细胞源外泌体对人脐静脉内皮细胞血管生成的影响 |
| 刘颖,尹园缘,邹巍莹,曹晖,宾东华 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南中医药大学, 湖南 长沙 410208) |
| 摘要: |
| 目的 探讨象皮生肌膏（Xiangpi Shengji Ointment, XPSJO）预处理的骨髓间充质干细胞源外泌体（XPSJO-Exos）对人脐静脉内皮细胞（human umbilical vein endothelial cell, HUVEC）血管生成的影响。方法 制备象皮生肌膏溶液，CCK-8法检测不同浓度象皮生肌膏（0、10、20、50、100、200 μg/mL）对骨髓间充质干细胞活力的影响；提取骨髓间充质干细胞中的外泌体（exosomes, Exos）和XPSJO-Exos，分别设为Exos组和XPSJO-Exos组，另设对照组（HUVEC），通过透射电子显微镜观察Exos的形态，采用粒径分析仪检测其粒径；Western blot检测Exos标志性蛋白TSG101、CD9和CD63的表达；采用PKH67标记Exos，观察其能否被HUVEC所摄取；采用CCK-8、划痕实验、小管形成实验、ELISA法检测Exos对HUVEC细胞增殖、迁移、小管形成和血管内皮生长因子（vascular endothelial growth factor, VEGF）浓度的影响；采用RT-qPCR检测血管生成相关基因ANG1、PDGF、bFGF、EGF的mRNA的表达。结果 10、20、50 μg/mL的XPSJO与0 μg/mL的XPSJO间细胞活力比较，差异均无统计学意义（P>0.05）。与0 μg/mL的XPSJO比较，100、200 μg/mL的XPSJO细胞活力显著降低（P<0.01）。在后续的实验中选用浓度50 μg/mL作为药物使用浓度。Exos组和XPSJO-Exos组均有呈球形、膜结合囊泡的典型Exos结构（大多数Exos粒径约为120 nm，且能被HUVEC摄取），并均检测到Exos标志性蛋白TSG101、CD9和CD63的表达。与对照组比较，Exos组和XPSJO-Exos组在72、96 h时细胞活力均显著增强（P<0.01），HUVEC小管数量、迁移率、VEGF浓度均显著增加或升高（P<0.05，P<0.01）。与Exos组比较，XPSJO-Exos组在72、96 h时细胞活力均显著增强（P<0.05），HUVEC小管数量、迁移率、VEGF浓度著增加或升高（P<0.05，P<0.01）。结论 XPSJO-Exos能促进HUVEC的增殖、迁移及小管形成，并促进VEGF的分泌。 |
| 关键词: 象皮生肌膏 外泌体 骨髓间充质干细胞 人脐静脉内皮细胞 血管生成 伤口愈合 |
| DOI：10.3969/j.issn.1674-070X.2023.02.011 |
| 投稿时间：2022-07-13 |
| 基金项目:国家自然科学基金项目（81603634）；湖南省中医药管理局重点课题（C2022019）；湖南省临床医疗技术创新引导项目（2021SK51416）；湖南中医药大学校级科研基金项目（2019XJJJ034）；湖南中医药大学中西医结合一流学科开放课题项目（2020ZXYJH05，2020ZXYJH57）；长沙市自然科学基金项目（Kq2202458）。 |
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| Effects of bone marrow mesenchymal stem cell-derived exosomes pretreated with Xiangpi Shengji Ointment on angiogenesis of human umbilical vein endothelial cell |
| LIU Ying,YIN Yuanyuan,ZOU Weiying,CAO Hui,BIN Donghua |
| (The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan University of Chinese Medicine, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To investigate the effects of bone marrow mesenchymal stem cell-derived exosomes (Exos) pretreated with Xiangpi Shengji Ointment (XPSJO) on the angiogenesis of human umbilical vein endothelial cell (HUVEC). Methods XPSJO solution was prepared, and CCK-8 method was used to detect the effects of different concentrations of XPSJO (0, 10, 20, 50, 100, 200 μg/mL) on the viability of bone marrow mesenchymal stem cell. Exos and XPSJO-Exos were extracted, set as Exos group and XPSJO-Exos group respectively, and HUVEC as control group. Meanwhile, the morphology of XPSJO-Exos was observed by transmission electron microscopy, the particle size of Exos was detected by particle size analyzer. The Exos marker proteins (TSG101, CD9 and CD63) were detected by Western blot, and Exos were labeled with PKH67 to observe whether they could be taken up by HUVEC. The CCK-8, scratch test, tubule formation test, and ELISA were respectively used to detect the effects of Exos on the proliferation, migration, tube formation and vascular endothelial growth factor (VEGF) expression of HUVEC, and the mRNA expression levels of angiogenesis-related genes (ANG1, PDGF, bFGF, and EGF) were detected by RT-qPCR. Results There was no significant difference in cell viability between 10, 20, 50 μg/mL XPSJO and 0 μg/mL XPSJO (P>0.05). Compared with the 0 μg/mL XPSJO, the cell viability in 100, 200 μg/mL XPSJO was significantly lower (P<0.01). 50 μg/mL concentration of XPSJO was used in the subsequent experiments. Both in the XPSJO-Exos group and XPSJO-Exos group, there were typical spherical Exos structures with membrane-bound vesicles (The particle size of most Exos was 120 nm, and Exos could be taken up by HUVEC), and the expression of Exos marker proteins (TSG101, CD9 and CD63) were detected. Compared with control group, the cell viability was significantly enhanced at 72 and 96 h both in the Exos group and XPSJO-Exos group (P<0.01), the number of tubules, migration rate and VEGF concentration of the HUVEC were significantly higher in the Exos group and XPSJO-Exos group (P<0.05, P<0.01). Compared with Exos group, the cell viability was significantly enhanced at 72 and 96 h in the XPSJO-Exos group (P<0.05), and the number of tubules, migration rate and VEGF concentration of the HUVEC were significantly higher in the XPSJO-Exos group (P<0.05, P<0.01). Conclusion XPSJO-Exos can promote the proliferation, migration and tubule formation of HUVEC and accelerate the secretion of VEGF. |
| Key words: Xiangpi Shengji Ointment exosomes bone marrow mesenchymal stem cell human umbilical vein endothelial cell angiogenesis wound healing |
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