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目的:比较重组25 kDa全长人釉原蛋白(recombinant human amelogenin,rhAm)和提取的猪釉基质蛋白(enamel matrix proteins,EMPs)对人牙周膜成纤维细胞(human periodontal ligament fibroblasts,HPDLF)和人包皮成纤维细胞(foreskin fibroblasts,HFF)生物学特性的影响,初步探讨rhAm促进牙周组织再生的可能机制。方法:选择BL21/pET28a-His-SUMO-rhAm表达系统,在适宜的条件下,经诱导表达和酶切纯化,得到25 kDa全长rhAm,采用SDS-PAGE和Western印迹鉴定,乙酸法提纯EMPs。体外培养HPDLF和HFF,采用不同浓度的rhAm和EMPs分别刺激HPDLF、HFF,观察并比较2种蛋白对细胞黏附、增殖和迁移能力的影响。采用SAS 5.0软件包对数据进行统计学分析。结果:10~20μg/mL rhAm能显著促进人牙周膜成纤维细胞和人包皮成纤维细胞的黏附、增殖和迁移(P<0.05),其作用效果与200μg/mL EMPs相似(P>0.05)。结论:25 kDa rhAm和EMPs对成纤维细胞具有相似的生物学作用。rhAm和EMPs可通过增强牙周膜成纤维细胞的生物学活性,从而促进牙周组织再生。
OBJECTIVE: To compare the effects of recombinant human amelogenin (25 kDa) and enamel matrix proteins (EMPs) on human periodontal ligament fibroblasts (HPDLF) and To explore the biological mechanism of human foreskin fibroblasts (HFF) and to explore the possible mechanism of rhAm promoting periodontal tissue regeneration. Methods: BL21 / pET28a-His-SUMO-rhAm expression system was selected, and the full-length 25 kDa rhAm was obtained by inducing expression and digestion purification under appropriate conditions. The recombinant protein was identified by SDS-PAGE and Western blot. EMPs were purified by acetic acid method. HPDLF and HFF were cultured in vitro. HPDLF and HFF were stimulated with different concentrations of rhAm and EMPs respectively. The effects of two proteins on cell adhesion, proliferation and migration were observed and compared. SAS 5.0 software package for statistical analysis of the data. Results: 10 ~ 20μg / mL rhAm could significantly promote the adhesion, proliferation and migration of human periodontal ligament fibroblasts and human foreskin fibroblasts (P <0.05), and the effect was similar to 200μg / mL EMPs (P> 0.05) . Conclusion: 25 kDa rhAm and EMPs have similar biological effects on fibroblasts. rhAm and EMPs promote periodontal tissue regeneration by enhancing the biological activity of periodontal ligament fibroblasts.