氟暴露对小鼠骨髓间充质干细胞增殖、凋亡及成骨分化的影响

Effects of fluoride exposure on proliferation,apoptosis and osteogenic differentiation of mouse bone marrow mesenchymalstemcells

目的探讨氟暴露对小鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)增殖、凋亡及成骨分化的影响。方法自C57BL/6小鼠(6~8周)股骨分离、培养得到BMSCs,取第3代细胞采用流式细胞术分析干细胞表面标志物。用氟终浓度分别为0.0、0.1、1.0、5.0、10.0、15.0、20.0、40.0 mg/L的培养基培养BMSCs,检测不同浓度氟对BMSCs细胞增殖(CCK8法)、凋亡(流式细胞术分析)、成骨分化能力[茜素红及碱性磷酸酶(ALP)染色]的影响;采用蛋白免疫印迹法(Western blot)检测凋亡相关蛋白[聚腺苷二磷酸核糖聚合酶(PARP)]、丝裂原活化蛋白激酶(MAPK)通路成员蛋白[细胞外调节蛋白激酶1/2(ERK1/2),c-Jun氨基末端激酶(JNK),p38及磷酸化ERK、JNK、p38(p-ERK、p-JNK、p-p38)],成骨分化相关蛋白[Runt相关转录因子2(Runx2)、ALP]与Wnt/β-连环蛋白(β-catenin)通路成员蛋白[糖原合成酶激酶-3β(GSK3β)、磷酸化GSK3β(p-GSK3β)及β-catenin]的表达水平;并采用免疫荧光染色分析p-GSK3β及β-catenin的表达情况;使用SP600125及DKK-1分别阻断MAPK和Wnt/β-catenin 2个信号通路后,分析细胞凋亡及成骨分化等的变化。结果成功分离、培养出小鼠BMSCs,流式细胞术分析显示,间充质干细胞表面标志分子CD73、CD90、CD105均呈阳性。各浓度组3个时间点(24、48、72 h)细胞增殖情况比较差异均有统计学意义(F=65.36、160.04、365.32,P均<0.001),各浓度组细胞早期凋亡情况(24 h)比较差异有统计学意义(F=214.04,P<0.001);与0.0 mg/L氟浓度组比较,15.0、20.0、40.0 mg/L氟浓度组细胞增殖水平均降低,10.0、15.0、20.0 mg/L氟浓度组细胞早期凋亡率均增高(P均<0.05)。15.0 mg/L氟处理细胞0~24 h,p-JNK/JNK比例在2、4、8、12、18、24 h时间点均高于0 min(P均<0.05);与单独氟处理组(15.0 mg/L)比较,SP600125阻断后细胞早期凋亡率降低(P<0.05),PARP及p-JNK蛋白表达水平均降低(P均<0.05)。成骨诱导后,与0.0 mg/L氟浓度组比较,0.1、1.0 mg/L氟浓度组ALP染色增强、矿化结节数量增多;且0.1、1.0 mg/L氟浓度组Runx2及ALP蛋白表达水平均较高(P均<0.05)。成骨诱导后,与0.0 mg/L氟浓度组比较,0.1、1.0 mg/L氟浓度组p-GSK3β/GSK3β比例及β-catenin蛋白表达水平均较高(P均<0.05);与单独氟处理组(1.0 mg/L)比较,DKK-1阻断后p-GSK3β及β-catenin蛋白表达水平均较低(P均<0.05),β-catenin入核减少,ALP染色减弱,矿化结节数目减少。结论高浓度氟(>10.0 mg/L)抑制BMSCs增殖、促进凋亡,而低浓度氟(0.1、1.0 mg/L)促进细胞成骨分化。MAPK/JNK通路与Wnt经典通路分别参与了上述细胞过程。

Objective To investigate the effects of fluoride exposure on proliferation,apoptosis and osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)in mice.Methods BMSCs were isolated and cultured from femur bone marrow of C57BL/6 mice(6-8 weeks).The cells in passage 3 were used to detect the surface markers of stem cells by flow cytometry.The cells were cultured in media with a final fluoride concentration of 0.0,0.1,1.0,5.0,10.0,15.0,20.0 and 40.0 mg/L,respectively.The effects of dfferent fluoride concentrations on BMSCs cell proliferation(CCK8 method),apoptosis(flow cytometry analysis),osteogenic differentiation ability[alizarin red and alkaline phosphatase(ALP)stainingl were detected.Western blot was applied to detect the levels of apoptosis-related proteins[poly ADP-ribose polymerase(PARP),mitogen-activated protein kinase(MAPK)pathway member proteins[extracellular regulated protein kinase 1/2(ERK1/2),c-Jun amino-terminal kinase(JNK),p38 and phosphorylated ERK,JNK,p38(p-ERK,p-JNK,p-p38)],osteogenic differentiation-related protein[Runt-related transcription factor 2(Runx2),ALP)and Wnt/β-catenin pathway member proteins[glycogen synthase kinase-3β(GSK3β),phosphorylated GSK3β(p-GSK3β)andβ-catenin),Immunocytofluorescense staining was applied to evaluate the expression levels of p-GSK3βandβ-catenin.The two pathways(MAPK and Wnt/β-catenin)were blocked by SP600125 and DKK-1,respectively,to testify their involvement in mechanisms of apoptosis and osteogenic differentiation.ResultsThe mouse BMSCs were successfully isolated and cultured.Flow cytometry analysis showed that the mesenchymal stem cell surface biomarkers(CD73,CD90 and CD105)were positively expressed.The comparison of cell proliferation at three time points(24,48 and 72 h)in each concentration group was statistically significant(F=65.36,160.04 and 365.32,P<0.001),and the comparison of early apoptosis(24 h)in each concentration group was statistically significant(F=214.04,P<0.001);compared with the 0.0 mg/L group,the cell proliferation in 15.0,20.0 and 40.0 mg/L groups decreased,and the early apoptosis rate in 10.0,15.0 and 20.0 mg/L groups increased(P<0.05).When cells were treated with 15.0 mg/L fluoride for 0-24 h,the p-JNK/JNK ratio was higher at 2,4,8,12,18 and 24 h compared with that at 0 min(P<0.05);compared with the fluoride group(15.0 mg/L),the early apoptosis rate of cells after SP600125 block decreased(P<0.05),and the protein expression levels of PARP and p-JNK decreased(P<0.05).After osteogenic induction,compared with the 0.0 mg/L group,in 0.1 and 1.0 mg/L groups ALP staining was enhanced and the number of calcified nodules increased,and the protein expression levels of Runx2 and ALP in the 0.1 and 1.0 mg/L groups were higher(P<0.05).After osteogenic induction,compared with the 0.0 mg/L group,the p-GSK3β/GSK3βratio andβ-catenin protein level were significantly higher in the 0.1 and 1.0 mg/L groups(P<0.05);and compared with the fluoride group(1.0 mg/L),addition of DKK-1 significantly decreased the protein expression levels of p-GSK3βandβ-catenin and reduced the nuclear entry ofβ-catenin,and ALP staining decreased and the number of calcified nodules decreased.ConclusionsHigh concentration of fluoride(>10.0 mg/L)inhibits the proliferation and promotes apoptosis of BMSCs,while low concentration of fluoride(0.1,1.0 mg/L)promotes osteogenic differentiation.The MAPK/JNK pathway and the classical Wnt pathway are involved in the above cellular processes,respectively.