氟中毒通过引起p16功能障碍抑制成骨细胞功能的机制

The mechanism by which fluorosis inhibits osteoblastic function by causing p16 dysfunction

目的探究氟中毒抑制成骨细胞功能的机制。方法筛选48只SD大鼠,分组:对照组(n=12),自来水(氟离子浓度<0.5 mg/L);高氟组(n=12),氟化钠(氟离子浓度33.00 mg/L);中氟组(n=12),氟化钠(氟离子浓度16.50 mg/L);低氟组(n=12),氟化钠(氟离子浓度8.25 mg/L)。对各组的突触体钙离子浓度进行测定,分离SD大鼠骨细胞测定不通实验组细胞活力、细胞周期进行测定;利用实时荧光定量PCR及western blot实验对p16基因及蛋白的表达量进行测定;对p16基因的3个片段进行染色质免疫沉淀分析。结果氟处理组[Ca2+]均升高;中-氟组细胞增殖能力增加(P<0.05)。随着氟浓度的逐渐增加,高-氟组细胞增殖能力下降;NaF可使S期细胞数量呈剂量依赖性增加(P<0.05),随着NaF浓度的升高,S期和G2/M期细胞比例增加,而G0/G1期细胞比例呈剂量依赖性下降。随着NaF浓度的增加,PI呈剂量依赖性增加(P<0.05);p16的mRNA水平(P<0.05)和蛋白水平(P<0.05)呈剂量依赖性下调。与空白组相比,在高-氟化钠治疗组,H3ac、H4ac、H3K4、H4K20的组蛋白脱乙酰作用更强(P<0.05)。结论氟中毒通过引起p16基因乙酰化抑制成骨细胞的功能。

Objective To explore the mechanism of fluorosis inhibiting osteoblasts function.Methods Forty-eight SD rats were divided into four groups according to fluoride concentration(FC)with 12 mice in each group:control group(tap water,FC<0.5 mg/L),high fluoride group(sodium fluoride,FC=33.00 mg/L),middle fluoride group(sodium fluoride,FC=16.50 mg/L),low fluoride group(sodium fluoride,FC=8.25 mg/L).The calcium concentration of synaptosomes in each group was measured and the osteocytes of SD rats were isolated to investigate the cell viability and cell cycle.The expression level of p16 gene and protein was measured by real-time fluorescent quantitative PCR and Western blot respectively.The three segments of p16 gene were analyzed by chro-matin immunoprecipitation.Results The level of[Ca2+]in the fluorine-treated group increased.The proliferation capacity of the medium-fluorine group increased significantly(P<0.05).However,with the increase of fluoride concentration,the proliferation of high-fluorine group cells decreased.NaF increased the number of cells in S phase in a dose-dependent manner(P<0.05).With the increase of NaF concentration,the proportion of cells in S phase and G2/M phase increased,whereas the proportion of cells in G0/G1 phase decreased in a dose-dependent manner.With the increase of NaF concentration,PI increased in a dose-dependent manner(P<0.05).The mRNA and protein levels of p16 were decreased in a dose-dependent manner(P<0.05).Compared with the blank group,h3ac,h4ac,H3K4 and h4k20 had stronger deacetylation effect(P<0.05).Conclusion Fluorosis inhibits the function of osteoblasts by inducing acetylation of p16 gene.