氟对成骨细胞MG-63 DNA甲基化的影响

Effect of fluoride on DNA methylation of osteoblast MG-63

目的观察不同剂量的氟(F^(-))对成骨细胞MG-63的毒性效应,及在毒性作用过程中对DNA甲基化效应及相关基因的影响。方法0.0、2.5、5、10和20 mg/L的F^(-)干预成骨细胞(MG-63)24、48、72和96 h后噻唑蓝比色法(MTT)检测细胞的增殖活力,Illumina甲基化850k芯片检测细胞的甲基化程度;实时荧光定量PCR(RT-PCR)、Western blotting检测染氟成骨细胞DNMT1、DNMT2、DNMT3a、Runx2和Osterix基因表达情况。结果≥72 h时,5 mg/L的F^(-)对成骨细胞MG-63增殖活力有明显的促进作用(P<0.05);≥72 h时,20 mg/L的F^(-)对成骨细胞MG-63增殖活力有明显的抑制作用(P<0.05);72 h时5 mg/L的F^(-)作用后DNA甲基化程度较0 mg/L组明显的降低(P<0.05);≥20 mg/L的F^(-)作用后DNA甲基化程度较0 mg/L组明显的升高((P<0.05)。5 mg/L的F^(-)干预成骨细胞MG-63后甲基化转移酶DNMT1、DNMT2、DNMT3a的mRNA和蛋白的表达量较0 mg/L组相比明显降低(P<0.05);成骨细胞特异性转录因子Runx2、Osterix的mRNA和蛋白的表达量较0 mg/L组相比明显增加(P<0.05);20 mg/L的F^(-)干预成骨细胞MG-63后甲基化转移酶DNMT1、DNMT2、DNMT3a的mRNA、蛋白表达量较0 mg/L组相比明显增加(P<0.05),成骨细胞特异性转录因子Runx2、Osterix的mRNA和蛋白的表达量较0 mg/L组相比明显降低(P<0.05)。结论低剂量的F^(-)对成骨MG-63的增殖具有促进作用,抑制了甲基化转移酶DNMT1、DNMT2、DNMT3a的mRNA和蛋白的表达量,降低了成骨细胞MG-63DNA甲基化程度,在一定程度上促进了成骨细胞特异性转录因子Runx2、Osterix的mRNA和蛋白的表达。高剂量的F^(-)对成骨MG-63的增殖起到抑制作用,但促进了甲基化转移酶DNMT1、DNMT2、DNMT3a的mRNA和蛋白的表达量,增加了成骨细胞MG-63DNA甲基化程度,在一定程度上抑制了成骨细胞特异性转录因子Runx2、Osterix的mRNA和蛋白的表达。

Objective To observe the toxic effects of different doses of fluorine(F^(-))on osteoblast MG-63,DNA methylation and related genes.Methods After 0.0,2.5,5,10 and 20 mg/L F^(-)intervention on osteoblasts(MG-63)for 24,48,72 and 96 h,the proliferation activity of osteoblasts was detected by thiazole Blue Colorimetry(MTT),Illumina methylation 850 k chip was used to detect the methylation degree of cells;Real time fluorescence quantitative PCR(RT-PCR)and Western blotting were used to detect the gene expression of DNMT1,DNMT2,DNMT3 a,Runx2 and Osterix in fluoride stained osteoblasts.Results≥72 h,5 mg/L F^(-)significantly promoted the proliferation of osteoblast MG-63(P<0.05);≥72 h,20 mg/L F^(-)significantly inhibited the proliferation of osteoblast MG-63(P<0.05);At 72 h,the degree of DNA methylation in 5 mg/L F^(-)group was significantly lower than that in 0 mg/L Group(P<0.05);The degree of DNA methylation was significantly higher in the group of≥20 mg/L than that in the group of 0 mg/L(P<0.05).The mRNA and protein expressions of methyltransferases DNMT1,DNMT2 and DNMT3 a in osteoblast MG-63 treated with 5 mg/L F^(-)were significantly lower than those in 0 mg/L Group(P<0.05);The mRNA and protein expressions of osteoblast specific transcription factors Runx2 andOsterix were significantly higher than those in 0 mg/L Group(P<0.05);The mRNA and protein expressions of methyltransferases DNMT1,DNMT2 and DNMT 3 a were significantly higher than those in the 0 mg/L Group(P<0.05),The mRNA and protein expressions of osteoblast specific transcription factors Runx2 and Osterix were significantly lower than those in 0 mg/L Group(P<0.05).Conclusion Low dose F^(-)can promote the proliferation of osteoblast MG-63,inhibit the expression of mRNA and protein of methyltransferase DNMT1,DNMT2 and DNMT3 a,reduce the degree of DNA methylation of osteoblast MG-63,and promote the expression of mRNA and protein of osteoblast specific transcription factors Runx2 and Osterix to a certain extent.High dose of Finhibited the proliferation of osteoblast MG-63,but promoted the expression of mRNA and protein of methyltransferases DNMT1,DNMT2 and DNMT3 a,increased the degree of DNA methylation of osteoblast MG-63,and inhibited the expression of mRNA and protein of osteoblast specific transcription factors Runx2 and Osterix to a certain extent.