背景:氟化物可以诱导成骨细胞的增殖和活化,导致氟骨症进展;然而,具体机制尚不清楚。
方法:使用MTT测定法检查细胞增殖。流式细胞术检测细胞周期分布。计算碱性磷酸酶(ALP)以评估骨形成和更新。使用MSP测定检查基因甲基化。使用qRT-PCR和Western印迹测定评估mRNA和蛋白质表达水平。
结果:低浓度NaF处理促进了成骨细胞细胞周期向S期的进展,从而以浓度依赖性方式加速细胞增殖和活化。此外,MGMT和MLH1基因的甲基化增加,它们的mRNA表达降低。此外,DNA甲基转移酶抑制剂5-AZA-dC抑制细胞活力,S期细胞数,低剂量NaF处理的成骨细胞中ALP活性和成骨相关蛋白水平。同时,5-AZA-dC抑制低剂量NaF处理的成骨细胞中MGMT和MLH1基因甲基化的增加,导致MGMT和MLH1mRNA表达增强。
结论:NaF处理导致成骨细胞DNA修复基因MGMT和MLH1甲基化,导致细胞增殖和活化,并导致氟骨症的发展。
BACKGROUND: Fluoride can induce the proliferation and activation of osteoblasts, resulting in skeletal fluorosis progression; however, the specific mechanism is unclear.
METHODS: Cell proliferation was examined using the MTT assay. Flow cytometry was performed to detect the cell cycle distribution. Alkaline phosphatase (ALP) was calculated to evaluate bone formation and turnover. Gene methylation was examined using the MSP assay. mRNA and protein expression levels were assessed using qRT-PCR and Western blot assays.
RESULTS: Low-concentration NaF treatment promoted the cell cycle progression of osteoblasts to S-phase, thus accelerating cell proliferation and activation in a concentration-dependent manner. In addition, the methylation of the MGMT and MLH1 genes was increased, and their mRNA expression was reduced. Furthermore, the DNA methyltransferase inhibitor 5-AZA-dC suppressed cell viability, cell number in S-phase, ALP activity and osteogenesis-related protein levels in osteoblasts treated with low doses of NaF. Meanwhile, 5-AZA-dC suppressed the increase in MGMT and MLH1 gene methylation in osteoblasts treated with low doses of NaF, leading to enhanced expression of MGMT and MLH1 mRNA.
CONCLUSIONS: NaF treatment led to methylation of the DNA repair genes MGMT and MLH1 in osteoblasts, resulting in cell proliferation and activation and causing the development of skeletal fluorosis.