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

作　　者：庄齐翔 夏一如 董家辰[1] 谢玉峰[1] 束蓉[1] ZHUANG Qixiang;XIA Yiru;DONG Jiachen;XIE Yufeng;SHU Rong(Department of Periodontology,Shanghai Ninth Peoples 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)

机构地区：[1]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

出　　处：《口腔医学》2022年第9期796-801,共6页Stomatology

基　　金：上海交通大学医学院附属第九人民医院交叉基金资助(JYJC201904)。

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、100.0μg/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,0.1μg/mL和10.0μg/mL rhAm可促进HUVEC的增殖(P<0.05),100.0μg/mL rhAm出现抑制HUVEC增殖的现象(P<0.01);划痕实验结果显示,10.0μg/mL rhAm可促进HUVEC迁移(P<0.05);实时定量PCR结果显示,10.0μg/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,10μg/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,10μg/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。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.

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 

分 类 号：R781.4[医药卫生—口腔医学]