中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (16): 2516-2521.doi: 10.12307/2022.250

• 材料力学及表面改性 material mechanics and surface modification • 上一篇    下一篇

材料表面化学和流体剪切力共同刺激对人脐静脉内皮细胞的影响

覃中杰1 ，陈思奇2，吴一民1，杨姣姣1，夏德林1，2   

  1. 1西南医科大学附属口腔医院口腔颌面外科， 四川省泸州市   646000；2西南医科大学附属医院整形外科，四川省泸州市   646000
  • 收稿日期:2020-08-31 修回日期:2020-09-04 接受日期:2020-11-11 出版日期:2022-06-08 发布日期:2021-12-22
  • 通讯作者: 夏德林，博士，教授，主任医师，西南医科大学附属口腔医院口腔颌面外科，四川省泸州市 646000；西南医科大学附属医院整形外科，国家临床重点建设专科， 四川省泸州市 646000
  • 作者简介:覃中杰，男，1995年生，重庆市人，汉族，西南医科大学在读硕士，主要从事血管内皮细胞成血管分化方面的研究。
  • 基金资助:
    四川省科技厅应用基础研究资助项目(2008JY0014)，项目负责人：夏德林；泸州市科技局项目[2013-S-48(8/30)]，项目参与人：夏德林

Effects of co-stimulation of material surface chemistry and fluid shear stress on human umbilical vein endothelial cells

Qin Zhongjie1, Chen Siqi2, Wu Yimin1, Yang Jiaojiao1, Xia Delin1, 2   

  1. 1Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Department of Plastic Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Received:2020-08-31 Revised:2020-09-04 Accepted:2020-11-11 Online:2022-06-08 Published:2021-12-22
  • Contact: Xia Delin, MD, Professor, Chief physician, Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Department of Plastic Surgery, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Qin Zhongjie, Master candidate, Department of Oral and Maxillofacial Surgery, Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    the Applied Basic Research Grant Project of Sichuan Science and Technology Department of Sichuan Province, No. 2008JY0014 (to XDL); Science and Technology Bureau Project of Luzhou, No. 2013-S-48(8/30) (to XDL)

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摘要：

文题释义：
自组装单分子膜：是分子通过化学键自发地在固-液或气-固界面形成的稳定有序膜，具有良好的结构特征。利用单分子层可以在材料表面修饰多种配体和功能基团，这种表面具有规则的分子结构，可以在复杂溶液中保持其原有的性质，通过固定在表面的化学基团可以将配体整合到分子层上，严格控制配体的密度和分子取向。
流体剪切力：是血液流动时与血管内壁间发生摩擦产生对于血管壁的作用力，方向平行于血管长轴，与流动方向相反，是血管内皮细胞受到的最主要的生物机械刺激。

背景：组织工程骨内缺乏完善的脉管系统，其中血管化是限制其广泛应用的关键，适当的基质化学和流体剪切力刺激能促进血管内皮细胞增殖分化和发挥功能，指导支架材料的设计制作。
目的：探究不同化学官能团和流体剪切力对人脐静脉内皮细胞的影响，以期找到材料化学与流体剪切力对人脐静脉内皮细胞调控的高效组合。
方法：在玻片表面分别制备以OH、CH3和NH2为末端化学官能团的自组装单分子膜，以空白玻片作为对照；将人脐静脉内皮细胞分别接种于4组玻片表面，培养15 min内检测ATP的释放量，培养1 h内检测NO的释放量，培养1 h后Western blotting检测内皮型一氧化氮合酶的表达，培养48 h后激光共聚焦显微镜观察黏着斑及F肌动蛋白的形成。将人脐静脉内皮细胞分别接种于4组玻片表面，当细胞融合至80%时给予加载1.5 N/m2 流体剪切力1 h(分别为OH-FSS组、CH3-FSS组、NH2-FSS组和FSS组)，加载15 min内检测ATP的释放量，加载1 h内检测NO的释放量，加载1 h后Western blotting检测内皮型一氧化氮合酶的表达。
结果与结论：①单纯化学刺激对人脐静脉内皮细胞的ATP、NO释放与一氧化氮合酶表达无明显影响，加载FSS刺激可增加ATP、NO的释放与内皮型一氧化氮合酶表达；当材料化学和流体剪切力同时作用时，人脐静脉内皮细胞释放的ATP和NO量及内皮型一氧化氮合酶表达与材料化学密切相关，其中NH2-FSS组中的ATP和NO释放量及内皮型一氧化氮合酶表达最高，其次为FSS组，CH3-FSS组和OH-FSS组最低；②激光共聚焦显微镜下可见，NH2组有大量黏着斑和细胞骨架蛋白F肌动蛋白，其次为对照组，CH3组和OH组最少；③结果表明，NH2官能团和流体剪切力共同作用时人脐静脉内皮细胞对剪切力刺激产生高效应答，其机制可能是在NH2表面上形成了最佳的黏着斑和F肌动蛋白。
缩略语：人脐静脉内皮细胞：human umbilical vein endothelial cells，HUVECs；流体剪切力：fluid shear stress，FSS

https://orcid.org/0000-0001-5849-3010 (覃中杰) 

中国组织工程研究杂志出版内容重点：生物材料；骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性；组织工程

关键词: 材料, 支架, 自组装单分子层, 材料表面化学, 流体剪应力, 人脐静脉内皮细胞, 蛋白

Abstract: BACKGROUND: There is a lack of perfect vascular system in tissue-engineered bone, among which vascularization is the key to limit its wide application. Appropriate matrix chemistry and fluid shear stress stimulation can promote the proliferation, differentiation and function of vascular endothelial cells, and guide the design and fabrication of scaffold materials.
OBJECTIVE: To explore the effects of different chemical functional groups and fluid shear stress (FSS) on human umbilical vein endothelial cells, in order to find an efficient combination of material chemistry and fluid shear stress on human umbilical vein endothelial cells. 
METHODS: Self assembled monolayers with OH, CH3 and NH2 as end functional groups were prepared as matrix materials and blank glass as control. Human umbilical vein endothelial cells were seeded on the surface of four groups of glasses. The release of ATP within 15 minutes after culture and NO within 1 hour after culture was detected. The expression of endothelial nitric oxide synthase protein was detected by western blotting. The formation of focal adhesion and F-actin was observed by laser confocal microscopy. Human umbilical vein endothelial cells were seeded on the surface of four groups of glasses. When the cells fused to 80%, they were loaded with 1.5 N/m2 FSS for 1 hour. ATP release was detected within 15 minutes after loading, and NO release was detected within 1 hour after loading. The expression of endothelial nitric oxide synthase was detected by western blotting after loading for 1 hour.
RESULTS AND CONCLUSION: (1) Chemical stimulation had no effect on the release of ATP or NO, but FSS could increase the release of ATP and NO, and the expression of endothelial nitric oxide synthase. When FSS and material chemistry acted simultaneously, the expression of ATP and NO was closely related to material chemistry. The release of ATP and NO and the expression of endothelial nitric oxide synthase were highest in NH2-FSS group, followed by FSS group, and those in the CH3-FSS and OH-FSS groups were lowest. (2) Laser confocal microscope showed that a large number of focal adhesions and cytoskeleton protein F-actin could be detected in the NH2 group, followed by control group; those were least in the CH3 and OH groups. (3) The results showed that the combination of NH2 functional group and FSS produced high-efficiency response to shear stress, and the mechanism may be the formation of optimal focal adhesion and F-actin on NH2 surface. 

Key words: material, scaffold, self-assembled monolayers, surface chemistry, fluid shear stress, human umbilical vein endothelial cells, protein

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