摘要
目的·探讨载重组人釉原蛋白(recombinant human amelogenin,rhAm)水凝胶缓释系统对人牙周膜成纤维细胞(human periodontal ligament fibroblasts, HPDLFs)增殖、迁移、黏附及成骨、成牙骨质分化能力的影响。方法·组织块法获取原代HPDLFs,传代培养后将第3～5代细胞分为对照组、蛋白组(20μg/mL rhAm)、载蛋白水凝胶组(含20μg/mL rhAm的PCLA-PEG-PCLA水凝胶)。以CCK-8法检测细胞增殖与生长曲线的变化,采用划痕实验及Transwell小室实验检测细胞迁移的效果,以细胞计数法及扫描电子显微镜(电镜)观察并检测细胞黏附的效果,采用实时荧光定量PCR技术测定ALP、Runx2、CEMP1和CAP mRNA的表达,观察成骨能力的改变。结果·载rhAm水凝胶缓释系统对HPDLFs的生长曲线没有显著影响,对其增殖有促进作用,第3日时差异有统计学意义(P<0.05)。划痕实验和Transwell小室实验中,蛋白组迁移最快,其次为载蛋白水凝胶组。载蛋白水凝胶对HPDLFs的黏附有促进作用,4 h时差异有统计学意义(P<0.05),扫描电镜下蛋白组与载蛋白水凝胶组的细胞伸展状态较好。HPDLFs经载蛋白水凝胶诱导培养后,ALP、Runx2、CEMP1、CAP mRNA表达在不同时间点有所上调。结论·载rhAm水凝胶缓释系统可显著促进HPDLFs的增殖和黏附,对其迁移无显著影响,对其成骨及成牙骨质能力均有显著促进作用。
Objective · To determine the effect of recombinant human amelogenin (rhAm)-loaded PCLA-PEG-PCLA hydrogels on cell proliferation, immigration, attachment and osteogenic differentiation of human periodontal ligament fibroblasts (HPDLFs). Methods · HPDLFs were obtained by tissue block method in vitro from extracted premolars and the 3rd-5th passages of HPDLFs were treated with DMEM medium (control group), 20 μg/mL rhAm (rhAm group) or rhAm-loaded PCLA-PEG-PCLA hydrogels (rhAm-loaded hydrogel group). Proliferation activity was measured by CCK-8, while cell migration was assayed both by wound-healing experiment in vitro and Transwell experiment. Cell attachment was measured by hemocytometer and observed by scanning electron microscope. Osteogenic differentiation was measured by real-time PCR, with ALP, Runx2, CEMP1 and CAP as the target genes. Results · RhAm-loaded PCLA-PEG-PCLA hydrogels had no significant effect on cell growth curve of HPDLFs, but promoted cell proliferation after 3 days (P<0.05). RhAm accelerated cell migration mostly both in wound-healing experiment and Transwell experiment, with rhAm-loaded hydrogels in the second place. RhAm-loaded hydrogels promoted cell attachment, and in the 4th hour the promotion was of statistic significance (P<0.05). Meanwhile cells of rhAm group and rhAm-loaded hydrogel group had a better stretch condition than control group under the scanning electron microscope. After culture with rhAm-loaded hydrogels, ALP, Runx2, CEMP1, and CAP mRNA were upregulated in different time points. Conclusion · Recombinant human amelogenin-loaded PCLA-PEG-PCLA hydrogels can significantly improve proliferation, attachment and osteogenic differentiation of HPDLFs, but has no effect on cell migration on a statistical scale.
作者
宁航
夏一如
董家辰
束蓉
NING Hang;XIA Yi-ru;DONG Jia-chen;SHU Rong(Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, Shanghai 200011, China)
出处
《上海交通大学学报（医学版）》
CAS
CSCD
北大核心
2019年第3期244-252,共9页
Journal of Shanghai Jiao tong University:Medical Science
基金
国家自然科学基金面上项目(81570977)~~
关键词
牙周组织再生
釉原蛋白
水凝胶
增殖
黏附
迁移
成骨分化
periodontal tissue regeneration
amelogenin
hydrogel
proliferation
attachment
migration
osteogenic differentiation
