摘要/Abstract
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（ 20 μg/mL rhAm）、载蛋白水凝胶组（含 20 μg/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。
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
Abstract:
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 obtainedtissue block method in vitro 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 measuredCCK-8, while cell migration was assayed bothwound-healing experiment in vitro and Transwell experiment. Cell attachment was measuredhemocytometer and observedscanning electron microscope. Osteogenic differentiation was measuredreal-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.
Key words: periodontal tissue regeneration, amelogenin, hydrogel, proliferation, attachment, migration, osteogenic differentiation
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