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北京大学学报（医学版） ›› 2021, Vol. 53 ›› Issue (2): 371-377. doi: 10.19723/j.issn.1671-167X.2021.02.023

• 论著 • 上一篇    下一篇

生物活性玻璃对人脐静脉血管内皮细胞增殖及成血管的作用

黄丽东,宫玮玉,董艳梅()   

  1. 北京大学口腔医学院·口腔医院,牙体牙髓科 国家口腔疾病临床医学研究中心 口腔数字化医疗技术和材料国家工程实验室 口腔数字医学北京市重点实验室, 北京 100081
  • 收稿日期:2019-02-13 出版日期:2021-04-18 发布日期:2021-04-21
  • 通讯作者: 董艳梅 E-mail:kqdongyanmei@bjmu.edu.cn
  • 基金资助:
    国家自然科学基金(81870753)

Effects of bioactive glass on proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells

HUANG Li-dong,GONG Wei-yu,DONG Yan-mei()   

  1. Department of Cariology and Endodontology, Peking University School and Hospital of Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Laboratory for Digital and Material Technology of Stomatology & Beijing Key Laboratory of Digital Stomatology, Beijing 100081, China
  • Received:2019-02-13 Online:2021-04-18 Published:2021-04-21
  • Contact: Yan-mei DONG E-mail:kqdongyanmei@bjmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(81870753)

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摘要：

目的: 研究以植酸为前驱体制备的生物活性玻璃(phytic acid derived bioactive P2O5-SiO2-CaO gel-glasses, PSC)对人脐静脉内皮细胞(human umbilical vein endothelial cells, HUVECs)增殖、分化及体外成血管的作用。方法: 用内皮细胞培养基(endothelial cell medium, ECM)按梯度浓度(0.01、0.1、1和2 g/L)分别制备PSC浸提液培养HUVECs,在第1、3、5、7、10天采用甲基噻唑基四唑[(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide, MTT]法对细胞的增殖能力进行检测,并与ECM培养的对照组HUVECs进行比较。根据增殖实验结果,选择最适宜HUVECs生长的PSC浸提液浓度用于后续实验。后续实验分为两组:实验组用PSC浸提液培养HUVECs(PSC组),对照组用ECM培养HUVECs(ECM组)。采用实时反转录聚合酶链反应(real-time reverse transcription-polymerase chain reaction, real-time RT-PCR)于第2、4、7天检测血管内皮生长因子(vascular endothelial growth factor, VEGF)、碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)的基因表达;通过小管形成实验观察第 4、10小时PSC浸提液对HUVECs形成小管的形态与数目的影响,并用Image J软件定量分析。结果: 用MTT法筛选最适生长浓度,结果提示0.1 g/L PSC浸提液对HUVECs的增殖促进作用最为显著,在第5、7天时细胞光密度值显著高于其他浓度实验组及对照组(P<0.05)。0.1 g/L PSC组可显著上调HUVECs成血管基因VEGF、bFGF的表达(P<0.05);第4天时,PSC组VEGF、bFGF的基因表达量分别是ECM组的1.59和1.45倍;第7天时,PSC组VEGF、bFGF的基因表达量分别是ECM组的1.98和1.37倍。在小管形成实验第10小时,PSC组小管的成熟度与密度优于ECM组,PSC组的小管数目(29.63±2.29)高于ECM组(20.13±2.36),差异有统计学意义(P<0.05)。结论: PSC具有促进HUVECs增殖、分化及体外成血管的作用。

关键词: 生物活性玻璃, 组织工程, 血管再生, 人脐静脉血管内皮细胞

Abstract:

Objective: To investigate the effects of phytic acid derived bioactive P2O5-SiO2-CaO gel-glasses (PSC) on the proliferation, differentiation and angiogenesis of human umbilical vein endothelial cells (HUVECs) in vitro. Methods: HUVECs were cultured in PSC extracts, which were prepared with endothelial cell medium (ECM) at a gradient concentration of 0.01, 0.1, 1 and 2 g/L. Cells cultured in ECM were used as the control. The effect of PSC on HUVECs proliferation was assessed on the 1st, 3rd, 5th, 7th and 10th days with (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide assay (MTT), and the optimum PSC concentration for HUVECs proliferation was used in the following experiments. The subsequent experiments were divided into two groups. The experimental group used PSC extracts to culture HUVECs (PSC group) and the control group used ECM to culture HUVECs (ECM group). Gene expression of angiogenic factors, vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), was detected on the 2nd, 4th and 7th days by real-time reverse transcription-polymerase chain reaction (real-time RT-PCR). The morphology and number of tubules formation were observed at the 4th and 10th hours. Image J software was used for counting and quantitative analysis. Results: The results of MTT assay showed that 0.1 g/L PSC group had the most significant effect on promoting HUVECs proliferation. The optical density values of 0.1 g/L PSC group on the 5th and 7th days were significantly higher than those of the other PSC groups and the control group (P<0.05). The result of real-time RT-PCR showed that 0.1 g/L PSC extract up-regulated the mRNA expression of VEGF and bFGF significantly (P<0.05). On the 4th day, the gene expressions of VEGF and bFGF in PSC group were 1.59 and 1.45 times higher than those in ECM group respectively, and on the 7th day, the gene levels of VEGF and bFGF in PSC group were 1.98 and 1.37 times higher than those in ECM group respectively. The tubule formation assay showed that the maturity and density of the tubules in 0.1 g/L PSC group was much better than that in the ECM group at the 10th hour. The quantitative analysis by Image J indicated that the tubules number in PSC group (29.63±2.29) was higher than in the ECM group (20.13±2.36), with statistical significance (P<0.05). Conclusion: PSC showed significant promoting effects on HUVECs’ proliferation, differentiation and angiogenesis in vitro.

Key words: Bioactive glass, Tissue engineering, Angiogenesis, Human umbilical vein endothelial cells

中图分类号: 

  • R782.12

表1

Real-time RT-PCR中成血管基因的引物序列"

Target genes Sequences of primers
VEGF Forward: 5'-GCAAGAAATCCCGTCCCT-3'
Reverse: 5'-TCGTTTAACTCAAGCTGCCTC-3'
bFGF Forward: 5'-AAGAGCGACCCTCACATCA-3'
Reverse: 5'-TCGTTTCAGTGCCACATACC-3'
GAPDH Forward: 5'-CAACGGATTTGGTCGTATTGG-3'
Reverse: 5'-GCAACAATATCCACTTTACCAGAGTTAA-3'

图1

PSC对HUVECs增殖的作用"

图2

PSC对HUVECs成血管基因VEGF、bFGF表达的作用"

图3

PSC对HUVECs形成小管的作用"

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ISSN 1671-167X
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