基因干扰免疫球蛋白结合蛋白对氟刺激成骨细胞作用的影响  

Effects of gene interference binding immunoglobulin protein on stimulation of osteoblast differentiation induced by fluoride

作　　者：赵志涛[1] 杨晨[1] 王岩[1] 李广生[1] 徐辉[1] 

机构地区：[1]吉林大学药学院地方病研究所病理研究室,长春130021

出　　处：《中华地方病学杂志》2015年第5期335-339,共5页Chinese Journal of Endemiology

基　　金：国家自然科学基金（81072249）;吉林大学白求恩计划（B）资助项目（2012222）;高等学校博士学科点专项科研基金（20130061110084）

摘　　要：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.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 f

关 键 词：氟化物 成骨细胞 RNA干扰 

分 类 号：R599.1[医药卫生—内科学]