国际口腔医学杂志 ›› 2019, Vol. 46 ›› Issue (2): 203-208.doi: 10.7518/gjkq.2019025

• 论著 • 上一篇    下一篇

精氨酰-甘氨酰-天冬氨酸多肽层层自组装修饰钛片对小鼠成骨样细胞MC3T3-E1的影响

刘菲1,张云涛2(),马向瑞3,张雅杰1,王云浩1   

  1. 1.滨州医学院口腔医学院 滨州 256600
    2.滨州医学院附属医院口腔修复科 滨州 256600
    3.滨州医学院附属医院口腔颌面外科 滨州 256600
  • 收稿日期:2018-07-04 修回日期:2018-10-11 出版日期:2019-03-01 发布日期:2019-03-15
  • 通讯作者: 张云涛 E-mail:zytao999@163.com
  • 作者简介:刘菲,住院医师,硕士,Email: 435792307@qq.com
  • 基金资助:
    山东省医药卫生科技发展计划项目(2017WS231)

Effect of the titanium surface immobilised by arginyl-glycyl-aspartate peptide self-assembling multilayers on mouse osteoblast-like cell MC3T3-E1

Fei Liu1,Yuntao Zhang2(),Xiangrui Ma3,Yajie Zhang1,Yunhao Wang1   

  1. 1. School of Stomatology, Binzhou Medical University, Binzhou 256600, China
    2.Dept. of Prosthodontics, Affiliated Hospital of Binzhou Medical University, Binzhou 256600, China
    3.Dept. of Oral and Maxillofacial Surgery, Affiliated Hospital of Binzhou Medical University, Binzhou 256600, China)
  • Received:2018-07-04 Revised:2018-10-11 Online:2019-03-01 Published:2019-03-15
  • Contact: Yuntao Zhang E-mail:zytao999@163.com
  • Supported by:
    This study was supported by Projects of Medical and Health Technology Development Program of Shandong Province(2017WS231)

摘要:

目的 研究层层自组装精氨酰-甘氨酰-天冬氨酸(RGD)多肽修饰钛片对小鼠成骨样细胞MC3T3-E1的影响。方法 将纯钛试件随机分为3组:纯钛组(PT组)、NaOH处理钛片组(NT组)、RGD修饰钛片组(RT组)。根据分组对纯钛试件进行相应表面处理,扫描电子显微镜(SEM)观察试件表面形貌。将MC3T3-E1细胞在试件表面培养,采用SEM、3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(MTT)法检测细胞的黏附和增殖能力,实时荧光定量聚合酶链式反应(RT-qPCR)法检测成骨细胞骨钙素、骨保护素mRNA的表达。结果 SEM观察到PT组试件表面存在均匀、较清晰的划痕结构,NT组试件表面出现微孔结构,RT组试件表面有类似小凸起结构,似网状。SEM观察到RT组细胞呈多边形且有大量丝状伪足,细胞伸展状态明显好于其余2组;MTT法显示RT组细胞的黏附率最高,无钛片对照组和PT组的细胞黏附率的差异无统计学意义,NT组细胞黏附率最低;RT组的细胞增殖率均高于其余3组(P<0.05),在1和3 d最为明显;RT-qPCR检测14 d时RT组骨钙素mRNA和骨保护素mRNA均高表达。结论 RGD多肽修饰钛片能促进小鼠成骨样细胞MC3T3-E1的黏附和增殖,并且能够上调骨钙素mRNA和骨保护素mRNA的表达。

关键词: 层层自组装, 精氨酰-甘氨酰-天冬氨酸多肽, 小鼠成骨样细胞MC3T3-E1

Abstract:

Objective This study aimed to investigate the effect of the titanium surface immobilised by arginyl-glycyl-aspartate (RGD) peptide self-assembling multilayers on the mouse osteoblast-like cell MC3T3-E1. Methods Metal specimens were randomly divided into three groups: pure titanium (PT) group, NaOH-treated titanium (NT) group and RGD-modified titanium (RT) group. The morphology of the specimens was inspected by scanning electron microscopy (SEM). MC3T3-E1 cells were cultured on the surface of specimens. The adhesion and proliferation of MC3T3-E1 cells were observed by SEM and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. The mRNA expression levels of osteocalcin and osteoprotegerin were quantified by real-time quantitative polymerase chain reaction. Results The sample surface of the PT group showed uniform and clear scratches, whereas that of the NT group showed microporous structure. Meanwhile, the surface structure of the RT group resembled the small bulges to a web surface. Cells adhering on RT disks exhibited active spreading and polygonous shape, with more filopodia. MTT assay showed that the adhesion rate in the RT group was the highest among the four groups; no considerable difference existed between the control group (withont titanium) and PT group. The cell proliferation rate of RT group was also the highest (P<0.05), especially at 1 and 3 days. In the RT group, osteocalcin mRNA and osteoprotegerin mRNA expression levels decreased more than those in the control group and PT group at 14 days (P<0.05). Conclusion RT can promote cell adhesion and proliferation, and increase osteocalcin mRNA and osteoprotegerin mRNA expression levels in MC3T3 cells.

Key words: layer-by-layer self-assembly, arginyl-glycyl-aspartate peptide, mouse osteoblast-like cell MC3T3-E1

中图分类号: 

  • Q813.1 +1

图 1

钛片的表面形态 SEM × 5 000 A:PT组钛片;B:NT组钛片;C:RT组钛片。"

表 1

不同钛片表面对小鼠成骨样细胞MC3T3-E1在不同时间点黏附的影响"

分组 1 h 4 h 12 h 24 h
对照组 0.035±0.006 0.044±0.006 0.092±0.006 0.129±0.004
PT组 0.036±0.005 0.045±0.005 0.095±0.005 0.134±0.005
NT组 0.028±0.005*# 0.035±0.005*# 0.075±0.004*# 0.100±0.009*#
RT组 0.049±0.003*#& 0.064±0.005*#& 0.104±0.005*#& 0.153±0.005*#&

图 2

钛片表面小鼠成骨样细胞MC3T3的黏附形态 SEM × 1 000=== A:PT组钛片;B:NT组钛片;C:RT组钛片。"

表 2

不同钛片表面对小鼠成骨样细胞MC3T3-E1增殖的影响"

分组 1 d 3 d 5 d 7 d
对照组 0.128±0.004 0.375±0.069 0.931±0.109 1.115±0.142
PT组 0.131±0.010 0.386±0.027 0.949±0.115 1.064±0.137
NT组 0.109±0.014*# 0.154±0.041*# 0.286±0.048*# 0.679±0.060*#
RT组 0.155±0.009*#& 0.544±0.118*#& 0.969±0.166& 1.138±0.082&

图 3

不同钛片表面对小鼠成骨样细胞MC3T3-E1骨钙素、骨保护素mRNA表达的影响 *相同时间点与PT组比较,P<0.05;#相同时间点与NT组比较,P<0.05。"

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