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目的 通过RNA干扰技术观察在不同剂量氟暴露条件下,成骨细胞内免疫球蛋白结合蛋白(binding immunoglobulin protein,BiP)表达减少对细胞成骨标志物和关键转录因子表达的影响.方法 采用小鼠成骨细胞(MC3T3-E1)作为细胞体外培养模型.取对数生长期MC3T3-E1细胞,接种子96孔板,培养24h后,加入含不同剂量氟离子[0.0(对照组)、0.1、1.0、2.0、4.0、8.0、16.0、20.0、32.0、64.0 mg/L]的培养液,于培养第1、3、7、14天采用Cell Counting Kit-8试剂盒检测细胞活性.不同剂量氟离子(2.0、8.0、20.0 mg/L)联合短片段RNA(small interfering RNA,siRNA)干扰BiP转染处理成骨细胞2d,利用实时定量PCR和蛋白杂交技术检测BiP基因mRNA和蛋白水平,同时检测成骨细胞内成骨标志物和关键转录因子的变化.结果 染氟第1、3、7、14天,不同剂量氟处理组间细胞活性比较,差异有统计学意义(F=46.7、118.6、214.6、325.6,P均＜0.05).染氟20.0 mg/L未转染组成骨细胞的BiP基因mRNA水平较对照组显著升高(11.22±3.25比7.94±1.31,P＜0.05).染氟2.0 mg/L未转染组成骨细胞碱性磷酸酶(alkaline phosphatase,ALP)mRNA表达较对照组显著提高(12.81±3.62比6.86±2.13,P＜ 0.01),而染氟20.0 mg/L未转染组细胞的ALP mRNA表达显著下降(0.89±0.17比6.87±2.14,P＜ 0.01).与相同剂量氟处理组比较,染氟2.0、8.0 mg/L联合转染BiP siRNA组细胞ALP mRNA表达显著下降(12.81±3.62比7.43±2.06;5.92±2.38比3.96±0.21,P均＜0.05);染氟2.0 mg/L联合BiP siRNA转染组细胞骨钙素(osteocalcin,OCN)mRNA表达下降(4.29±0.99比1.29±0.86,P＜ 0.01).染氟2.0 mg/L未转染组细胞成骨细胞关键转录因子Runx2(Runt-related transcription factor 2)表达高于未转染对照组(6.61±0.48比2.66±0.39,P＜0.05).成骨细胞转染BiP siRNA后各加氟组Runx2表达较转染对照组显著下降(1.13±0.22比4.81±1.03,3.20±0.66比4.81±1.03,0.02±0.02比4.81±1.03,P均＜0.01).染氟2.0、20.0 mg/L细胞转染BiP siRNA后的Runx2 mRNA表达较相同剂量氟处理组明显降低(1.13±0.22比6.61±0.48;0.02±0.02比1.50±0.38,P均＜0.01).Runx2下游因子osterix的表达变化与染氟联合BiP siRNA转染细胞的Runx2改变一致.结论 BiP在氟刺激成骨作用中主要影响成骨标志物和关键转录因子的表达,而不是直接参与氟刺激成骨细胞作用.“,”Objective To observe the effect of RNA interference binding immunoglobulin protein (BiP) on expression of bone markers and keytranscription factors in osteoblast exposed to fluoride.Methods MC3T3-E1 cells were used as osteoblast model in vitro.The cell viability was test with cell counting Kit-8 after cells were administrated with varying concentrations of fluoride [0.0 (control),0.1,1.0,2.0,4.0,8.0,16.0,20.0,32.0 and 64.0 mg/L] for different duration.Cells transfected with small interfering RNA (siRNA) BiP were exposed to fluoride (2.0,8.0 and 20.0 mg/L) for 2 days.Real-time PCR and Western blotting technique were used to determine the gene and protein levels of BiP.Meantime,the mRNA expression of bone markers and key transcription factors was investigated by real-time PCR.Results The difference of all viability in fluoride-dose groups was statistically significant exposed for 1,3,7 and 14 days (F =46.7,118.6,214.6,325.6,all P ＜ 0.05).Expression of BiP significantly increased in cells exposed to 20.0 mg/L compared to that of control (11.22 ± 3.25 vs.7.94 ± 1.31,P ＜ 0.05).The expression of alkaline phosphatase (ALP) elevated in cells exposed to 2.0 mg/L of fluoride (12.81 ± 3.62 vs.6.86 ± 2.13,P ＜ 0.01);conversely,it significantly reduced in cells exposed to 20.0 mg/L of fluoride (0.89 ± 0.17 vs.6.87 ± 2.14,P ＜ 0.01).Cells transfected with siRNA BiP significantly decreased the ALP expression in cells exposed to fluoride compared to that of cells only exposed to the same concentration of fluorine (12.81 ± 3.62 vs.7.43 ± 2.06;5.92 ± 2.38 vs.3.96 ± 0.21,all P ＜ 0.05).Cells transfected with siRNA BiP and administrated with 2.0 mg/L significantly reduced the osteocalcin expression (4.29 ± 0.99 vs.1.29 ± 0.86,P ＜ 0.01).Similarly,expression of runt-related transcription factor 2 (Runx2) significantly increased in cells exposed to 2.0 mg/L.However,expression of Runx2 significantly decreased in cells transfected with siRNA BiP and administrated with fluoride.Cells transfected with siRNA BiP significantly decreased the Runx2 expression in cells exposed to 2.0 mg/L and 20.0 mg/L compared to that of cells only exposed to the same concentration of fluorine (1.13 ± 0.22 vs.6.61 ± 0.48;0.02 ± 0.02 vs.1.50 ± 0.38,all P ＜ 0.01).As the downstream of Runx2,the expression of osterix in cells treated with different concentrations of fluoride was similar to that of Runx2.Conclusion BiP is not directly involved in the process of osteoblast differentiation induced by fluoride;instead,it affects the expression of bone markers and key transcription factors in osteoblast exposed to fluoride.