补骨脂素对炎性环境下人牙周膜干细胞成骨分化的影响及机制

Effect and mechanism of psoralen on osteogenic differentiation of human periodontal ligament stem cells in inflammatory environment

目的探究补骨脂素对人牙周膜干细胞(hPDLSCs)成骨分化作用及可能机制。方法利用酶消化法和有限稀释法体外分离并纯化hPDLSCs,流式细胞术鉴定细胞表型,成骨及成脂诱导检测细胞多向分化能力;取第3代hPDLSCs并分为对照组、肿瘤坏死因子-α(TNF-α)诱导组(10 ng/mL)、补骨脂素低(TNF-α10 ng/mL+补骨脂素2μmol/L)、中(TNF-α10 ng/mL+补骨脂素6μmol/L)、高(TNF-α10 ng/mL+补骨脂素18μmol/L)剂量组,各组均利用成骨诱导培养液培养。7 d后,碱性磷酸酶(ALP)检测试剂盒检测ALP活性;RT-qPCR技术检测Runt相关转录因子2(Runx2)、锌指结构转录因子(Osx)、骨桥蛋白(OPN)、骨钙素(OCN)mRNA相对表达水平;Western blot技术检测Wnt3a、β-连环蛋白(β-catenin)、糖原合成酶激酶3β(GSK-3β)、磷酸化糖原合成酶激酶3β(p-GSK-3β)、组织蛋白酶K(CTSK)蛋白相对表达水平;21 d后,茜素红染色及比色分析法检测钙沉积情况。结果经细胞表型及分化能力鉴定,确定分离培养的细胞为hPDLSCs;经成骨化及炎性环境诱导后,与对照组比较,TNF-α诱导组、补骨脂素3个治疗组中ALP活性、矿化钙沉积吸光度值、Runx2、Osx、OPN及OCN mRNA相对表达水平、Wnt3a、β-catenin及p-GSK-3β蛋白相对表达水平降低,CTSK蛋白相对表达水平升高(P<0.05);与TNF-α诱导组比较,补骨脂素3个治疗组中除CTSK蛋白相对表达水平降低外,其他上述指标水平均升高(P<0.05);补骨脂素作用效果呈剂量依赖性(P<0.05)。结论补骨脂素可促进炎性环境下hPDLSCs成骨分化,其作用机制可能与激活Wnt/β-catenin轴下调CTSK蛋白表达有关。

Objective To explore the effect and possible mechanism of psoralen on osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs).Methods hPDLSCs were isolated and purified in vitro by enzyme digestion and limited dilution method.The cell phenotype was identified by flow cytometry,and the ability of multidirectional differentiation was detected by osteogenic and adipogenic induction.The third generation hPDLSCs were taken and divided into the control group,tumor necrosis factor-α(TNF-α)induction group(10 ng/mL)and low,medium and high doses of psoralen groups.All groups were with osteogenic induction medium.The three doses of psoralen groups were contained 10 ng/mL TNF-αand 2,6 and 18μmol/L psoralen,respectively.After 7 days,alkaline phosphatase(ALP)activity was detected by ALP test kit,and the expression of runt related transcription factor 2(Runx2),zinc finger structure transcription factor(Osx),osteopontin(OPN)and osteocalcin(OCN)mRNA was detected by RT-qPCR.Western blot was used to detect the expression of Wnt3a,β-catenin,glycogen synthase kinase 3β(GSK-3β),phosphorylated glycogen synthase kinase 3β(p-GSK-3β),cathepsin K(CTSK)protein.After 21 days,alizarin red staining and colorimetric analysis were used to detect calcium deposition.Results Through the identification of cell phenotype and differentiation ability,the isolated and cultured cells were identified as hPDLSCs.After osteogenesis and inflammatory environment induction,compared with the control group,ALP activity,absorbance of mineralized calcium deposition,the expression levels of Runx2,Osx,OPN and OCN mRNA,and the expression levels of Wnt3a,β-catenin and p-GSK-3βprotein were decreased and the expression level of CTSK protein was increased in TNF-αinduction group and psoralen treatment group(P<0.05).Compared with TNF-αinduction group,the expression level of CTSK protein was decreased in the three psoralen-treated groups,but the levels of other above-mentioned indexes were increased in a dose-dependent manner(P<0.05).Conclusion Psoralen can promote the osteogenic differentiation of hPDLSCs in an inflammatory environment,which may be related to the downregulation of CTSK expression by activating Wnt/β-catenin axis.