摘要
目的构建人原代成骨细胞体外氟中毒模型，观察不同剂量氟化钠（NaF）对人成骨细胞细胞周期蛋白D1（CyelinD1）、细胞周期蛋白依赖性激酶4（CDK4）基因组蛋白乙酰化水平的影响，探讨氟骨症发生发展的分子机制。方法收集骨科外伤手术健康人（车祸）骨组织，通过酶消化法分离人原代成骨细胞，经鉴定后作为实验对象。以0、125、250、500及1000μmol／L NaF处理细胞72h。定量染色质免疫共沉淀技术检测CyelinD1、CD1（4基因转录调控区CHIP1区域及编码区CHIP2区域（对照区）组蛋白H3K9、H3K14、H4K12、H4K16乙酰化水平。结果①分别以0、125、250、500及1000μmol／L NaF处理细胞72h后，CyclinD1基因转录调控区CHIP1区域组蛋白H3K9乙酰化水平分别为1．152±0．104、1．174±0．187、1．090±0．176、1．170±0．197、1．147±0．097，组间比较差异无统计学意义（F=0．524，P〉0．05）；H3K14乙酰化水平分别为1．495±0．117、1．4654-0．069、1．470±0．187、1．760±1．089、1．341±0．443，组间比较差异无统计学意义（F=0．841，P〉0．05）；H4K12乙酰化水平分别为1．239±0．286、0．702±0．063、0．765±0．370、1．011±0．321、1．319±0．026，组间比较差异无统计学意义（F=2．329，P〉0．05）：H4K16乙酰化水平分别为1．452±0．217、1．621±0．165、1．462±0．090、1．510±0．146、1．564±0．154，组间比较差异无统计学意义（F=0．123，P〉0．05）。②CDK4基因转录调控区CHIPl区域组蛋白H3K9乙酰化水平分别为1．472±0．163、1．580±0．161、1．585±0．132、1．451±0．136、1．560±0．039，组间比较差异无统计学意义（F=0．461，P〉0．05）；H3K14乙酰化水平分别为0．919±0．149、0．900±0．059、0．911±0．162、0．663±0．049、0．841±0．122，组间比较差异无统计学意义（F=0．974，P〉0．05）；H4K12乙酰化水平分别为0．456±0．142、0．911±0．126、0．969±0．185，1．110±0．146、0．931±0，141，组间比较差异无统计学意义（F=5．459，P〉0．05）：H4K16乙酰化水平分别为1．315±0．083、1．374±0．153、1．423±0．055、1．300±0．132、1．385±0．696，组间比较差异无统计学意义（F=1．663，P〉0．05）。（3）CyclinD1、CDK4基因编码区CHIP2区域组蛋白H3K9、H3K14、H4K12、H4K16乙酰化水平，各NaF处理组间比较差异均无统计学意义（F=0．392、0．823、0．999、0．397，0．705、0．049、1．065、0．196，P均〉0．05）。结论在氟处理的人原代成骨细胞中，未观察到细胞周期调控基因CyelinD1、CDK4转录调控区组蛋白乙酰化参与其表达调控。
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 ehromatin immuno-precipitation （Q-CHIP）. Results （1）After 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）. （2）The 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）. （3）The 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.
出处
《中华地方病学杂志》
CAS
CSCD
北大核心
2018年第1期13-18,共6页
Chinese Journal of Endemiology
基金
国家自然科学基金（81260418）
贵州省优秀科技教育人才省长基金（黔省专合字[2011]54号）
