釉基质衍生物对人牙周膜干细胞成骨分化的影响

Effects of enamel matrix derivative on proliferation and osteogenic differentiation of human periodontal ligament stem cells

目的研究釉基质衍生物对人牙周膜干细胞增殖和成骨分化的影响并探究其可能的机制。方法原代培养人牙周膜干细胞,经过流式鉴定后选取第3代细胞进行实验。采用CCK-8试剂盒检测不同浓度(0、20、50、100 mg·L^-1)的釉基质衍生物对人牙周膜干细胞增殖的影响;实时荧光定量聚合酶链式反应(qRT-PCR)检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞成骨分化的影响;通过Trichrome染色和Von Kosa’s染色检测不同浓度(0、20、50、100 mg·L^-1)釉基质衍生物对人牙周膜干细胞胶原合成和矿化结节形成的影响;不同浓度釉基质衍生物和DDK1作用人牙周膜干细胞之后,通过Western blot和qRT-PCR检测β-连环蛋白、RunX2、CaMKⅡ及NLK表达情况。结果釉基质衍生物对人牙周膜干细胞的增殖具有明显的促进作用,并呈现剂量和时间依赖性;釉基质衍生物处理人牙周膜干细胞之后,矿化结节形成和胶原合成显著增多,骨钙素、Ⅰ型胶原、RunX2的表达明显增多;另外,釉基质衍生物处理能显著增加β-连环蛋白、RunX2、CaMKⅡ和NLK的表达,且该作用可被DDK1抑制。结论釉基质衍生物对体外培养的人牙周膜干细胞有促进增殖和成骨分化的作用,其作用可能是通过Wnt/β-连环蛋白实现的。

Objective To investigate the effects of enamel matrix derivative(EMD)on the proliferation and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)and the potential mechanism.Methods The primary cultured hPDLSCs were identified by flow cytometry,and the third generation cells were selected for experiments.The cell counting kit-8 assay was performed to detect the effect of different EMD concentrations(0,20,50 and 100 mg·L^-1)on the proliferation of hPDLSCs.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to verify the effect of EMD on the osteogenic differentiation of hPDLSCs in different concentrations(0,20,50 and 100 mg·L^-1).The effect of different EMD concentrations(0,20,50 and 100 mg·L^-1)on collagen synthesis and mineralised nodule formation of hPDLSCs were detected through trichrome and Von Kosa’s staining.Western blotting and qRT-PCR were performed to detectβ-catenin,RunX2,CaMKⅡand NLK expressions after different concentrations of EMD and DDK1 were applied to hPDLSCs.Results EMD significantly promoted the proliferation of hPDLSCs,which increased with the increase in concentration and stimulation time of EMD.After EMD treatment with hPDLSCs,the formation of mineralised nodules and collagen synthesis increased significantly.Furthermore,the expressions of osteocalcin,collagenⅠand RunX2 were elevated.Moreover,the expressions ofβ-catenin,RunX2,CaMKⅡand NLK were significantly increased with EMD treatment and inhibited by DDK1.Conclusion EMD can promote the proliferation and osteogenic differentiation of hPDLSCs in vitro through the Wnt/β-catenin signalling pathway.