氟化钠对人原代成骨细胞p21基因组蛋白乙酰化及表达的影响
Effects of Sodium Fluoride on Histone Acetylation and Expression of p21 Gene in Human Primary Osteoblasts
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摘要:
目的 观察氟化钠对人原代成骨细胞
p21 基因组蛋白乙酰化水平、mRNA转录及蛋白表达的影响，为阐释氟骨症发生分子机制提供依据。方法 以0、125、250、500、1 000 μmol/L氟化钠处理人原代成骨细胞72 h，定量染色质免疫共沉淀技术检测
p21 基因转录调控区ChIP1区域及编码区ChIP2区域组蛋白乙酰化水平，实时荧光定量PCR法检测p21 基因mRNA转录水平，免疫印迹法检测P21蛋白表达。结果 氟化钠处理的0、125、250、500、1 000 μmol/L浓度组中，
p21 基因转录调控区ChIP1区域组蛋白H3K9乙酰化水平分别为1.998±0.085、1.644±0.162、1.381±0.069、1.280±0.129、1.040±0.270，差异有统计学意义（F =15.757，P < 0.05）；组蛋白H3K14乙酰化水平分别为1.344±0.068、1.248±0.070、1.140±0.090、1.040±0.116、0.770±0.059，差异有统计学意义（F =21.284，P < 0.05）；组蛋白H4K16乙酰化水平分别为1.330±0.084、1.251±0.085、1.087±0.044、0.854±0.070、0.788±0.051，差异有统计学意义（F =36.429，P < 0.05）。不同浓度染氟组中，编码区ChIP2区域组蛋白H3K9、H3K14、H4K16乙酰化水平差异均无统计学意义（F =0.084、0.206、0.500，均P >0.05）。p21 基因mRNA转录水平分别为2.65±0.35、1.93±0.48、0.88±0.17、0.82±0.09、0.67±0.16，差异有统计学意义（F =3.90，P < 0.05）；P21蛋白表达水平分别为1.62±0.06、1.02±0.08、0.84±0.07、0.26±0.05、0.07±0.03，差异亦有统计学意义（F =281.58，P < 0.05）。结论 氟可致人原代成骨细胞
p21 基因转录调控区组蛋白乙酰化水平降低，抑制p21 基因mRNA转录及蛋白表达，可能是氟致人成骨细胞增殖活跃的重要分子机制之一。Abstract:Objective To observe the effects of sodium fluoride on the levels of histone acetylation, mRNA transcription, and protein expression of
p21 gene in human primary osteoblasts and provide evidence for a potential molecular mechanism of sk eletal fluorosis.Methods Primary osteoblasts were treated with 0, 125, 250, 500, and 1 000 μmol/L sodium fluoride for 72 h. The levels of histone acetylation in transcription regulatory region (ChIP1 region) and coding region (ChIP2 region) of
p21 gene were detected by quantitative chromatin immuno precipitation. The levels ofp21 mRNA transcription were detected by real-time quantitative PCR. The expression levels of P21 protein were detected by Western blot.Results After human osteoblasts were tre ated with 0, 125, 250, 500, and 1 000 μmol/L sodium fluoride, in ChIP1 transcription regulatory region of
p21 gene, the levels of histone acetylation of H3K9 were 1.998±0.085, 1.644±0.162, 1.381±0.069, 1.280±0.129, and 1.040±0.270, respectively (F =15.757,P < 0.05); the levels of histone acetylation of H3K14 were 1.344±0.068, 1.248±0.070, 1.140±0.090, 1.040±0.116, and 0.770±0.059, respectively (F =21.284,P < 0.05); the levels of histone acetylation of H4K16 were 1.330±0.084, 1.251±0.085, 1.087±0.044, 0.854±0.070, and 0.788±0.051, respectively (F =36.429,P < 0.05). Among the groups treated with different concentrations of fluoride, the levels of histone acetylation of H3K9, H3K14, and H4K16 in ChIP2 coding region were not statistically different (F =0.084, 0.206, and 0.500, allPs >0.05). The levels ofp21 mRNA transcription were 2.65±0.35, 1.93±0.48, 0.88±0.17, 0.82±0.09, and 0.67±0.16, respectively (F =3.90,P < 0.05). The levels of P21 protein expression were 1.62±0.06, 1.02±0.08, 0.84±0.07, 0.26±0.05, and 0.07±0.03, respectively (F =281.58,P < 0.05).Conclusion Fluoride could reduce the levels of histone acetylation of
p21 gene transcription regulatory region in human primary osteoblasts, then suppress the expression ofp21 mRNA transcription and the protein, which might be one of the important molecular mechanisms of active cell proliferation in skeletal fluorosis.
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