补肾益气活血方及其单味药对人牙周膜干细胞增殖分化的影响

Effects of Bushen Yiqi Huoxue Recipe and Its Single Drug on Proliferation and Differentiation of Human Periodontal Ligament Stem Cells

目的:探讨自拟补肾益气活血方及其单味药当归、补骨脂对体外培养的人牙周膜干细胞(hPDLSCs)增殖和相关成骨基因表达的影响。方法:分离培养得到hPDLSCs,选取第3代hPDLSCs,细胞增殖试剂盒(CCK-8)检测不同浓度(0 g/mL、1×10^(-7) g/mL、1×10^(-5) g/mL、1×10^(-3) g/mL)补肾益气活血方和当归、补骨脂对hPDLSCs增殖的影响,并确定最佳作用浓度和最佳作用时间;通过定量聚合酶链式反应(qPCR)检测Runt相关转录因子2(Runx2)、碱性磷酸酶(ALP)、骨桥蛋白(OPN)、骨钙蛋白(OCN)相关成骨基因的表达水平,通过茜素红染色观察细胞成骨分化情况。结果:与0 g/mL相比,补肾益气活血方各浓度在第一天和第三天时均能显著促进细胞增殖(P<0.05),且浓度为1×10^(-5) g/mL在第三天、第五天时效果均最为显著(P<0.05);当归同样在浓度为1×10^(-5) g/mL、第三天及第五天时效果均最为显著(P<0.05);补骨脂则仅在第一天时能显著促进细胞增殖(P<0.05)。与空白对照组比较,1×10^(-5) g/mL的补肾益气活血方、当归、补骨脂均能显著提升成骨相关Runx2、OCN、OPN、ALP的表达(P<0.05),且补肾益气活血方的上调效果最为显著。茜素红染色检测显示,补肾益气活血方可增加矿化结节,促进作用最为显著。结论:补肾益气活血方可促进hPDLSCs的增殖和骨向分化,在治疗慢性牙周炎中有望发挥更大作用。

Objective:To investigate the effects of self-designed Bushen Yiqi Huoxue Recipe and its single drug Angelica sinensis and Psoralea corylifolia on the proliferation and osteogenic gene expression of human periodontal ligament stem cells(hPDLSCs)in vitro.Methods:hPDLSCs were isolated and cultured.The third generation hPDLSCs were selected.Cell proliferation Kit(CCK-8)was used to detect the effects of different concentrations(0 g/mL,1×10^(-7) g/mL,1×10^(-5) g/mL,1×10^(-3) g/mL)of Bushen Yiqi Huoxue Recipe,Angelica sinensis and Psoralea corylifolia on the proliferation of hPDLSCs,and the determine the best action concentration and the best action time were determined.The expression levels of Runt related transcription factor 2(Runx2),alkaline phosphatase(ALP),osteopontin(OPN)and Osteocalcin(OCN)related osteogenic genes were detected by quantitative polymerase chain reaction(qPCR),and the osteogenic differentiation was observed by alizarin red staining.Results:Compared with 0 g/mL,Bushen Yiqi Huoxue Recipe could significantly promote cell proliferation on the first day and the third day(P<0.05),and the concentration of 1×10^(-5) g/mL had the most significant effect on the third day and the fifth day(P<0.05).Angelica sinensis on the concentration of 1×10^(-5) g/mL also had the most significant effect on the third day and the fifth day(P<0.05).Psoralea corylifolia could significantly promote cell proliferation only on the first day(P<0.05).Compared with the blank control group,1×10^(-5) g/mL of Bushen Yiqi Huoxue Recipe,Angelica sinensis and Psoralea corylifolia could significantly increase the expression of Runx2,OCN,OPN and ALP(P<0.05),and the up regulation effect of Bushen Yiqi Huoxue Recipe was the most significant.Alizarin red staining showed that Bushen Yiqi Huoxue Recipe could increase mineralized nodules,and the most significant effect was promoting.Conclusion:Bushen Yiqi Huoxue Recipe can promote the proliferation and osteogenic differentiation of hPDLSCs,which is expected to play a greater role in the treatment of chronic periodontitis.