甲基结合蛋白1对人脐静脉内皮细胞迁移、增殖以及血管形成的影响

doi:10.12122/j.issn.1673-4254.2026.07.12

南方医科大学学报 ›› 2026, Vol. 46 ›› Issue (7): 1585-1592.doi: 10.12122/j.issn.1673-4254.2026.07.12

• • 上一篇

甲基结合蛋白1对人脐静脉内皮细胞迁移、增殖以及血管形成的影响

但昱彤¹(), 张庭辅¹, 刘红梅¹^,²^,³^,⁴(), 张鹏¹^,²^,³()

^1.贵州医科大学，组织工程与干细胞实验中心，贵州贵阳 550004
^2.贵州医科大学，贵州生物制造实验室，贵州贵阳 550004
^3.贵州医科大学，功能核酸生物药研究重点实验室，贵州贵阳 550004
^4.中国科学院广州生物医药与健康研究院，广东广州 510530

收稿日期:2025-12-15 出版日期:2026-07-20 发布日期:2026-07-20
通讯作者: 刘红梅,张鹏 E-mail:danyutong2023@163.com;lhm2017851002@163.com;peng12zhang@gmc.edu.cn
作者简介:但昱彤，在读硕士研究生，E-mail: danyutong2023@163.com
基金资助:
国家自然科学基金(32000601);贵州省教育厅高校科技创新团队，黔教计[2023]068号;广州市科技计划项目(2023A04J0720);贵州医科大学高层次人才科研启动基金项目(校博合J字（2024）065号)

MBD1 knockdown inhibits proliferation, migration and angiogenesis of human umbilical vein endothelial cells in vitro

Yutong DAN¹(), Tingfu ZHANG¹, Hongmei LIU¹^,²^,³^,⁴(), Peng ZHANG¹^,²^,³()

^1.Center for Tissue Engineering and Stem Cell Research, Guizhou Medical University, Guiyang 550004, China
^2.Guizhou Biomanufacturing Laboratory, Guizhou Medical University, Guiyang 550004, China
^3.Key Laboratory of Functional Nucleic Acids-Based Biopharmaceutical Research, Guizhou Medical University, Guiyang 550004, China
^4.Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China

Received:2025-12-15 Online:2026-07-20 Published:2026-07-20
Contact: Hongmei LIU, Peng ZHANG E-mail:danyutong2023@163.com;lhm2017851002@163.com;peng12zhang@gmc.edu.cn
Supported by:
National Natural Science Foundation of China(32000601)

摘要/Abstract

摘要：

目的探讨甲基结合蛋白1（MBD1）对人脐静脉内皮细胞（HUVECs）增殖、迁移以及血管形成的影响。方法设置MBD1敲低（sh-MBD1）组和对照（sh-NC）组。使用靶向人MBD1基因shRNA LV3（H1/GFP&Puro）-MBD1-HOMO-270的慢病毒液和携带Scramble shRNA的慢病毒液LV3（H1/GFP&Puro）-NC感染HUVECs细胞，使用嘌呤霉素筛选感染成功的细胞。通过实时定量PCR（RT-qPCR）以及蛋白质印迹检测MBD1基因的沉默效率。通过CCK-8和克隆形成实验检测细胞增殖情况，流式细胞术检测细胞周期改变，细胞划痕实验以及Transwell实验检测细胞迁移速度，血管形成实验检测MBD1对HUVECs的血管形成能力的影响，免疫荧光检测MBD1对HUVECs中血管内皮生长因子A（VEGFA）表达的影响。结果成功构建MBD1敲低的HUVECs；与sh-NC组相比，sh-MBD1组细胞的增殖能力、增殖指数以及迁移能力显著降低（P<0.0001）；sh-MBD1组细胞体外生成的血管总长度、血管闭合环路以及血管分支点均低于sh-NC组细胞（P<0.01）；sh-MBD1组细胞中VEGFA表达量较对照细胞显著降低（P<0.0001）。结论 MBD1促进人脐静脉内皮细胞增殖、迁移和血管形成，且促进作用可能通过调控VEGFA的表达实现。

关键词: 血管形成, 甲基结合蛋白1, 人脐静脉内皮细胞, 血管内皮生长因子A, 细胞增殖, 细胞迁移

Abstract:

Objective To investigate the effects of methyl-CpG binding domain protein 1 (MBD1) on proliferation, migration, and angiogenesis in human umbilical vein endothelial cells (HUVECs). Method Cultured HUVECs were infected with a lentivirus carrying sh-MBD1 or a scramble shRNA (sh-NC), and the successfully infected cells were screened using puromycin. RT-qPCR and Western blotting were used to assess MBD1 gene silencing efficiency. The changes in proliferation, cell cycle, and migration of the infected cells were assessed using CCK-8 and colony formation assays, flow cytometry, scratch assay, and Transwell assay. The impact of MBD1 knockdown on angiogenic capacity of HUVECs was evaluated using angiogenesis assay. Immunofluorescence staining was used to investigate the expression of vascular endothelial growth factor A (VEGFA) in the infected HUVECs. Result Compared with the sh-NC cells, HUVECs infected with the lentivirus carrying sh-MBD1 exhibited significantly reduced proliferation capacity and migration ability. The total length of vessels formed in vitro, closed vascular loops, and the number of branching points were all lower in sh-MBD1 cells than in sh-NC cells. VEGFA expression was significantly reduced in sh-MBD1 cells compared to the sh-NC cells. Conclusion MBD1 promotes proliferation, migration, and angiogenesis of HUVECs possibly by regulating the expression of VEGFA.

Key words: angiogenesis, methyl-CpG-binding domain protein 1, human umbilical vein endothelial cells, vascular endothelial growth factor A, cell proliferation, cell migration

但昱彤, 张庭辅, 刘红梅, 张鹏. 甲基结合蛋白1对人脐静脉内皮细胞迁移、增殖以及血管形成的影响[J]. 南方医科大学学报, 2026, 46(7): 1585-1592.

Yutong DAN, Tingfu ZHANG, Hongmei LIU, Peng ZHANG. MBD1 knockdown inhibits proliferation, migration and angiogenesis of human umbilical vein endothelial cells in vitro[J]. Journal of Southern Medical University, 2026, 46(7): 1585-1592.

图/表 6

图1 sh-MBD1组以及sh-NC组细胞的构建与验证

Fig.1 Construction and validation of human umbilical vein endothelial cells (HUVECs) with MBD1 knockdown. A: Fluorescence microscopy of HUVECs 48 h after lentivirus infection (Original magnification: ×50). B: qPCR detection of MBD1 mRNA expression levels in both groups (n=3). C: Western blotting for assessing interference efficiency and statistics of MBD1 protein expression levels (n=4). **P<0.01, ***P<0.001 vs sh-NC group.

图2 敲低MBD1对HUVECs迁移能力的影响

Fig.2 Effect of MBD1 knockdown on migration capacity of HUVECs. A: Observation and statistical analysis of scratch healing rates in HUVECs at 12 h (n=15). B: HUVECs scratch healing rate and statistics at 12 h (n=14). C: Transwell assay for assessing migration capacity of the infected cells (n=7). Scale bar=100 μm. **P<0.01, ****P<0.0001 vs sh-NC group.

图3 敲低MBD1对HUVECs增殖能力的影响

Fig.3 Effect of MBD1 knockdown on proliferation of HUVECs. A: Results of clonogenic assay of the infected HUVECs (n=6). B: CCK-8 assay for assessing cell viability of the infected cells (n=8). C: Proliferation curves of the infected cells based on CCK-8 assays (n=3). *P<0.01, ***P<0.001, ****P<0.0001 vs sh-NC group.

图4 敲低MBD1对HUVECs细胞周期的影响

Fig.4 Effect of MBD1 knockdown on cell cycle of HUVECs. A: Distribution and proportion of cells in G0/G1 phase, S phase, and G2/M phase in the two groups. B: Statistical analysis of the proliferation index in the two groups (n=7). ****P<0.0001 vs sh-NC group.

图5 敲低MBD1对HUVECs成管能力的影响

Fig.5 Effect of MBD1 knockdown on angiogenic capacity of HUVECs. A: Schematic diagram of tubule formation experimental workflow. B: Photographs of the cells at 2, 4, and 6 h post-tubing induction. C: Photographs of both cell groups after 24 h of tubule formation observed under a fluorescence microscope. D: Statistical analysis of total tubule length, meshes, and branching points at 6 h in both groups (n=6). Scale bar=100 μm. **P<0.01 vs sh-NC group.

图6 免疫荧光检测HUVECs中VEGFA表达

Fig.6 Immunofluorescence staining for detection of VEGFA Expression in HUVECs. A: VEGFA fluorescence expression (Scale bar=100 μm). B: Statistical analysis of VEGFA fluorescence intensity in the two cell groups. ****P<0.0001 vs sh-NC group.

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甲基结合蛋白1对人脐静脉内皮细胞迁移、增殖以及血管形成的影响

MBD1 knockdown inhibits proliferation, migration and angiogenesis of human umbilical vein endothelial cells in vitro

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