重组人釉原蛋白促进人脐静脉内皮细胞成血管分化作用的研究

doi:10.13591/j.cnki.kqyx.2022.09.006

口腔医学 ›› 2022, Vol. 42 ›› Issue (9): 796-801.doi: 10.13591/j.cnki.kqyx.2022.09.006

重组人釉原蛋白促进人脐静脉内皮细胞成血管分化作用的研究

庄齐翔, 夏一如, 董家辰, 谢玉峰, 束蓉

上海交通大学医学院附属第九人民医院牙周病科,上海交通大学口腔医学院,国家口腔医学中心,国家口腔疾病临床医学研究中心,上海市口腔医学重点实验室,上海(200011)

修回日期:2022-03-09 出版日期:2022-09-28 发布日期:2022-09-20
通讯作者: 束蓉 Tel:(021)53315510 E-mail:shurong1977@163.com
基金资助:
上海交通大学医学院附属第九人民医院交叉基金资助(JYJC201904)

Effect of recombinant human amelogenin on angiogenesis of human umbilical vein endothelial cell in vitro

ZHUANG Qixiang, XIA Yiru, DONG Jiachen, XIE Yufeng, SHU Rong

Department of Periodontology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai 200011, China

Revised:2022-03-09 Online:2022-09-28 Published:2022-09-20

摘要/Abstract

摘要： 目的观察重组人釉原蛋白(recombinant human amelogenin,rhAm)对体外培养的人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)成血管作用的影响。方法体外培养HUVEC,分别将浓度为0.1、10.0、50.0、100.0 μg/mL的rhAm作用于细胞,与对照组比较,采用MTT法观察HUVEC增殖,通过划痕实验观察HUVEC迁移,运用实时定量PCR和免疫印迹检测rhAm对HUVEC表达成血管相关基因及膜蛋白的影响,经由体外小管生成实验观察HUVEC小管样结构。结果 MTT结果显示,0.1 μg/mL和10.0 μg/mL rhAm可促进HUVEC的增殖(P＜0.05),100.0 μg/mL rhAm出现抑制HUVEC增殖的现象(P＜0.01);划痕实验结果显示,10.0 μg/mL rhAm可促进HUVEC迁移(P＜0.05);实时定量PCR结果显示,10.0 μg/mL rhAm可上调细胞间黏附分子-1(intercellular cell adhesion molecule-1,ICAM-1)(P＜0.01)和E选择素(E-selectin)(P＜0.05)的mRNA表达,血管内皮生长因子受体(vascular endothelial growth factor receptor,VEGFR)-1、VEGFR-2 mRNA表达有上调趋势但不存在统计学差异;免疫印迹实验结果显示,10 μg/mL rhAm可上调ICAM-1(P＜0.01)的蛋白表达,E-selectin、VEGFR-1和VEGFR-2的蛋白表达有上调趋势但不存在统计学差异;体外小管生成实验显示,10 μg/mL rhAm可诱导HUVEC排列成小管样结构。结论低浓度的rhAm能促进HUVEC的增殖及迁移和成血管相关基因及蛋白的表达,诱导小管样结构生成,提示rhAm在牙周组织再生过程中可能存在成血管作用。

关键词: 重组人釉原蛋白, 成血管作用, 内皮细胞, 牙周组织再生

Abstract: Objective To investigate the effects of recombinant human amelogenin(rhAm) on angiogenesis of human umbilical vein endothelial cell(HUVEC) in vitro. Methods HUVEC were cultured in vitro. The effect of different concentrations of rhAm(0.1, 10.0, 50.0, 100.0 μg/mL rhAm) were compared with control group. The proliferation of HUVEC was investigated via MTT assay. The migration of HUVEC was investigated via scratch assay. Expression of angiogenesis-related genes and proteins were measured via real-time PCR and western blotting. The angiogenic structure formation of HUVEC was investigated via tube formation assay. Results The proliferation of HUVEC was stimulated by rhAm at concentration of 0.1 μg/mL and 10.0 μg/mL(P＜0.05), while inhibited at concentration of 100.0 μg/mL (P＜0.01). At concentration of 10.0 μg/mL, rhAm stimulated HUVEC migration in scratch assay(P＜0.01). According to the result of real-time PCR, the mRNA expression of intercellular cell adhesion molecule-1 (ICAM-1)(P＜ 0.01) and E-selectin (P＜0.05) were increased by rhAm at concentration of 10.0 μg/mL; the mRNA expression of vascular endothelial growth factor receptor(VEGFR)-1 and VEGFR-2 had a trend of up-regulation but no statistical difference. According to the result of western blotting, the protein expression of ICAM-1(P＜0.01)was increased by rhAm at concentration of 10.0 μg/mL, while the protein expression of VEGFR-1, VEGFR-2 and E-selectin had a trend of up-regulation but no statistical difference. RhAm induced the formation of angiogenic structure in vitro. Conclusion Results of the present in vitro study show the potential influence of rhAm on the angiogenic activity of HUVEC at a lower concentration, which may play an important role in periodontal tissue regeneration.

Key words: recombinant human amelogenin, angiogenesis, endo-thelial cell, periodontal tissue regeneration

中图分类号:

R781.4

庄齐翔, 夏一如, 董家辰, 谢玉峰, 束蓉. 重组人釉原蛋白促进人脐静脉内皮细胞成血管分化作用的研究[J]. 口腔医学, 2022, 42(9): 796-801.

ZHUANG Qixiang, XIA Yiru, DONG Jiachen, XIE Yufeng, SHU Rong. Effect of recombinant human amelogenin on angiogenesis of human umbilical vein endothelial cell in vitro[J]. Stomatology, 2022, 42(9): 796-801.

参考文献

[1] Vaquette C, Pilipchuk SP, Bartold PM, et al. Tissue engineered constructs for periodontal regeneration:Current status and future perspectives[J]. Adv Healthc Mater, 2018, 7(21):e1800457.
[2] Félétou M. The endothelium:Part 1:Multiple functions of the endothelial cells—focus on endothelium-derived vasoactive mediators[M]. San Rafael: Morgan & Claypool life sciences, 2011.
[3] Nowak-Sliwinska P, Alitalo K, Allen E, et al. Consensus guidelines for the use and interpretation of angiogenesis assays[J]. Angiogenesis, 2018, 21(3):425-532.
[4] Sturtzel C. Endothelialcells[J]. Adv Exp Med Biol, 2017, 1003(9):71-91.
[5] Miron RJ, Sculean A, Cochran DL, et al. Twenty years of enamel matrix derivative:The past, the present and the future[J]. J Clin Periodontol, 2016, 43(8):668-683.
[6] 林智恺, 束蓉, 宋忠臣. 重组釉原蛋白的研究进展[J]. 牙体牙髓牙周病学杂志, 2011, 21(1):48-51.
[7] 陈发明, 高丽娜, 陈芳. 牙周再生治疗现状和进展[J]. 口腔疾病防治, 2019, 27(1):9-16.
[8] Tavelli L, McGuire MK, Zucchelli G, et al. Biologics-based regenerative technologies for periodontal soft tissue engineering[J]. J Periodontol, 2020, 91(2):147-154.
[9] 林智恺, 束蓉, 程岚, 等. 重组全长人釉原蛋白对人成纤维细胞黏附、增殖、迁移的影响[J]. 上海口腔医学, 2013, 22(1):7-12.
[10] Hakki SS, Bozkurt SB, Türkay E, et al. Recombinant amelogenin regulates the bioactivity of mouse cementoblasts in vitro[J]. Int J Oral Sci, 2018, 10(2):15.
[11] 廖蔚文, 宋忠臣, 束蓉, 等. 炎症微环境下重组人釉原蛋白对人牙周膜成纤维细胞炎症因子表达的影响[J]. 上海交通大学学报(医学版), 2016, 36(1):33-37.
[12] Lamalice L, le Boeuf F, Huot J. Endothelial cell migration during angiogenesis[J]. Circ Res, 2007, 100(6):782-794.
[13] Amin HD, Olsen I, Knowles J, et al. A tyrosine-rich amelogenin peptide promotes neovasculogenesis in vitro and ex vivo[J]. Acta Biomater, 2014, 10(5):1930-1939.
[14] Andrukhov O, Gemperli AC, Tang Y, et al. Effect of different enamel matrix derivative proteins on behavior and differentiation of endothelial cells[J]. Dent Mater, 2015, 31(7):822-832.
[15] Jonke E, Gemperli AC, Zhang TW, et al. Effect of tyrosine-rich amelogenin peptide on behavior and differentiation of endothelial cells[J]. Clin Oral Investig, 2016, 20(8):2275-2284.
[16] 廖蔚文, 宋忠臣, 束蓉. 炎症微环境对牙周组织再生的影响[J]. 口腔医学, 2015, 35(9):786-788.
[17] Bui TM, Wiesolek HL, Sumagin R. ICAM-1:A master regulator of cellular responses in inflammation, injury resolution, and tumorigenesis[J]. J Leukoc Biol, 2020, 108(3):787-799.
[18] Zhong L, Simard MJ, Huot J. Endothelial microRNAs regulating the NF-κB pathway and cell adhesion molecules during inflammation[J]. FASEB J, 2018, 32(8):4070-4084.
[19] Melincovici CS, Boşca AB, Şuşman S, et al. Vascular endothelial growth factor (VEGF)-key factor in normal and pathological angiogenesis[J]. Rom J MorpholEmbryol, 2018, 59(2):455-467.
[20] Afacan B, Öztürk VÖ, PaşalıÇ, et al. Gingival crevicular fluid and salivary HIF-1α, VEGF, and TNF-α levels in periodontal health and disease[J]. J Periodontol, 2019, 90(7):788-797.

重组人釉原蛋白促进人脐静脉内皮细胞成血管分化作用的研究

Effect of recombinant human amelogenin on angiogenesis of human umbilical vein endothelial cell in vitro

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	肖涛, 贺一家, 朱瑶萍, 郝峰瑶, 陈艳, 王志勇. 口腔鳞癌VEGF诱导树突状细胞致免疫耐受的机制研究[J]. 口腔医学, 2023, 43(3): 204-211.
[2]	王一玉, 黄佳萍, 丁佩惠, 董研. 联合生物材料的牙源性间充质干细胞进行牙周组织再生的研究进展[J]. 口腔医学, 2023, 43(3): 261-266.
[3]	孙典, 肖轶婧, 沈铭. 环状RNA-BIRC6促进血管新生的作用研究[J]. 口腔医学, 2022, 42(6): 508-514.
[4]	苏珊, 朱文卿, 邱憬. 载锌纳米线修饰钛表面的血管内皮细胞生物学行为研究[J]. 口腔医学, 2022, 42(5): 404-410.
[5]	徐东升许兵陈铁楼冯旭周以钧方以群吴织芬. 高压氧暴露富血小板纤维蛋白对调控血小板内皮细胞粘附分子和血管内皮生长因子分泌的时间效应关系研究[J]. , 2018, 38(9): 769-773.
[6]	刘岩李维善苏红影邵伟然王泽. 牙龈卟啉单胞菌脂多糖对人脐静脉内皮细胞MMP-9表达影响的研究[J]. , 2017, 37(5): 394-397.
[7]	廖悦宋忠臣. 介孔材料在牙周再生中的应用进展[J]. , 2017, 37(3): 258-261.
[8]	李杨徐增琦詹甜甜高洁程杰吴煜农叶金海. 包载血管内皮生长因子的乙二胺/聚己内酯微囊的制备及其生物活性的研究[J]. , 2016, 36(9): 778-783.
[9]	廖蔚文宋忠臣束蓉. 炎症微环境对牙周组织再生的影响[J]. , 2015, 35(9): 786-788.
[10]	李文军贾惠杰葛颂. Ⅰ、ⅣfimA型牙龈卟啉单胞菌对内皮细胞与平滑肌细胞共培养体系产生内皮素-1及一氧化氮的影响[J]. , 2015, 35(7): 525-531.
[11]	王芳葛少华. 甲状旁腺激素的成骨机制及其对颅颌面和牙周组织再生的研究进展[J]. , 2015, 35(12): 1076-1079.
[12]	孙文娟唐倩黄南楠. Wnt信号通路与牙周膜[J]. , 2014, 34(1): 65-67.
[13]	邓　辉,徐　静,余　溢,欧阳玉玲,吴亚菲. 牙龈卟啉单胞菌侵入对人血管内皮细胞分泌MCP-1影响的研究[J]. , 2012, 32(6): 325-327,343.
[14]	鲁红,田宇,吴织芬. 松质骨基质作为细胞载体应用于牙周组织工程的实验研究[J]. , 2012, 32(5): 270-273.
[15]	徐静；刘芬；刘芳强；余溢；欧阳玉玲；邓辉. 牙龈卟啉单胞菌感染对人脐静脉内皮细胞表达IL-8影响的研究[J]. , 2012, 32(4): 193-195,222.