ABSTRACT
Objective To establish an model of fluorosis with human primary osteoblasts in vitro and to detect the influences of different doses of sodium fluoride (NaF) on histone acetylation of CyclinD1,cyclindependent kinases 4 (CDK4) gene in human osteoblasts,then to explore the molecular mechanism of skeletal fluorosis from epigenetic perspective of the cell cycle regulation related genes.Methods Human primary osteoblasts from bone tissues of trauma surgery healthy people (car accident) were isolated by enzyme digestion and identified.The osteoblasts were treated with 0,125,250,500 and 1 000 μmol/L NaF for 72 h.The level of histone acetylation (H3K9,H3K14,H4K12,H4K16) in the transcription regulatory region (ChIP1 region) and in the coding region (ChIP2 region) of CyclinD1 and CDK4 genes were detected by quantitative chmmatin immuno-precipitation (Q-ChIP).Results ①After human osteoblasts were treated with 0,125,250,500 and 1 000 μmol/L NaF,respectively,the levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of CyclinD1 gene were 1.152 ± 0.104,1.174 ± 0.187,1.090 ± 0.176,1.170 ± 0.197 and 1.147 ± 0.097,respectively,the differences were not statistically significant (F =0.524,P ＞ 0.05);the average levels of histone acetylation of H3K14 were 1.495 ± 0.117,1.465 ± 0.069,1.470 ± 0.187,1.760 ± 1.089 and 1.341 ± 0.443,the differences were not statistically significant (F =0.841,P ＞ 0.05);the levels of histone acetylation of H4K12 were 1.239 ± 0.286,0.702 ± 0.063,0.765 ± 0.370,1.011 ± 0.321 and 1.319 ± 0.026,the differences were not statistically significant (F =2.329,P ＞ 0.05);the levels of histone acetylation of H4K16 were 1.452 ± 0.217,1.621 ± 0.165,1.462 ±0.090,1.510 ± 0.146 and 1.564 ± 0.154,the differences were not statistically significant (F =0.123,P ＞ 0.05).②The levels of histone acetylation of H3K9 in ChIP1 transcription regulatory region of CDK4 were 1.472 ± 0.163,1.580 ± 0.161,1.585 ± 0.132,1.451 ± 0.136 and 1.560 ± 0.039,the differences were not statistically significant (F =0.461,P ＞ 0.05);the levels of histone acetylation of H3K14 were 0.919 ± 0.149,0.900 ± 0.059,0.911 ±0.162,0.663 ± 0.049 and 0.841 ± 0.122,the differences were not statistically significant (F =0.974,P ＞ 0.05);the levels of histone acetylation of H4K12 were 0.456 ± 0.142,0.911 ± 0.126,0.969 ± 0.185,1.110 ± 0.146 and 0.931 ± 0.141,the differences were not statistically significant (F=5.459,P ＞ 0.05);the levels of histone acetylation of H4K16 were 1.315 ± 0.083,1.374 ± 0.153,1.423 ± 0.055,1.300 ± 0.132 and 1.385 ± 0.696,the differences were not statistically significant (F =1.663,P ＞ 0.05).③The differences of histone acetylation levels of H3K9,H3K14,H4K12 and H4K16 in ChIP2 coding region of CyclinD1 and CDK4 genes were not statistically significant between NaF treatment groups (F =0.392,0.823,0.999,0.397,0.705,0.049,1.065,0.196,P ＞ 0.05).Conclusion The histone acetylation of CyclinD1 and CDK4 may not be involved in the transcriptional regulation in human primary osteoblasts treated with fluoride.