染氟成骨细胞中凋亡相关microRNA筛选和鉴定

Fluorosis （drinking water type） caused by excessive fluoride intake, can cause joint deformation, rigidity pain, dysfunction, which is a serious damage to the residents' health, but even now the pathogenic mechanism is unclear. Previous studies had found that high concentrations of fluoride could cause osteoblast endoplasmic reticulum and mitochondria swelling, and apoptosis. Some abnormal apoptosis signaling molecules expression also were found. Due to fluorine can interfere a series gene expression in the osteoblasts, and the miRNA can regulate osteoblast apoptosis, thus we put forward the scientific hypothesis: fluoride induced apoptosis of osteoblasts, it may be associated with changing the miRNA's expression. through typical clinical cases, animal experiments, and fluorine poisoning cells model in vitro respectively, this project are to study the characteristics of apoptosis in osteoblast caused by high concentration fluoride. Pathway of apoptosis will be as the breakthrough point, we plan to study significant expressions of apoptosis signaling molecules and microRNAs in osteoblast treated with excessive fluoride. correlation between them will also be explored. The mechanism of signaling molecules of apoptosis regulated by microRNAs will be explored to uncover the role of miRNA in apoptosis induced by fluoride. From the angle of apoptosis regulated by the miRNA, this project 's aim is to explore pathogenic mechanism of fluorosis, by illustrating the mechanism of miRNA regulating osteoblast apoptosis signal transduction, and explain how apoptosis appear in the osteoblast in skeletal fluorosis. It also can provide new ideas and directions for new treatment for fluorosis.

饮水型氟中毒由摄入过量氟所致，引起患者关节变形，僵直疼痛，功能障碍，严重危害居民健康，但是致病机制未明。前期研究发现高浓度氟可引起成骨细胞内质网和线粒体肿胀，细胞凋亡，同时发现部分凋亡信号分子表达异常。由于氟能广泛干预成骨细胞内一系列基因表达，而miRNA能调控成骨细胞凋亡，因此提出科学假设：氟对成骨细胞的致凋亡作用，可能与改变miRNA表达有关。本项目分别通过临床病例、氟中毒细胞模型及动物实验，围绕高浓度氟致使成骨细胞凋亡的特点，以凋亡通路为切入点，研究过量氟作用下成骨细胞凋亡过程中表达显著变化的凋亡信号分子和miRNA以二者的相关性，探索miRNA调控凋亡信号分子的机制，明确其在成骨细胞染氟后诱导凋亡的作用。本项目从凋亡受miRNA调控角度来探讨氟中毒致病机制，通过阐明miRNA调控成骨细胞凋亡信号的机理，解释氟骨症中骨细胞凋亡现象，为寻找氟中毒治疗手段提供新的思路和方向。

以人成骨肉瘤细胞系 Saos-2 建立体外染氟模型。用流式细胞仪检测干预 24 h 后的线粒体膜电位；用 PCR芯片检测84个与凋亡相关基因；对差异表达基因用免疫印记法予以验证。同时用 20 mg/L NaF和40 mg/L NaF分别处理细胞 24/48 h后用 PCR芯片法测定凋亡相关 miRNA 的表达情况。原代培养小鼠成骨细胞建立染氟模型,用20 mg/L NaF和40 mg/L NaF分别处理12/24小时后再对miRNA进行测序筛选差异表达的miRNA。差异表达的miRNA利用双荧光素酶报告基因验证其潜在靶基因靶向关系。采用小鼠饮水染毒法建立氟中毒模型，测定骨组织中Caspase14的表达。患者标本检测miRNA的表达。研究结果表明，当氟化钠质量浓度为 20，40，80 mg/L 时，成骨细胞线粒体膜电位分别为27.0%，28.8%，38.6%；PCR芯片检测发现13个基因表达上调，15个基因表达下调。免疫印记显示Bim、Caspase 9、Caspase14、BCL2、BAX表达随氟化钠剂量增高而增强；Caspase 3在5 mg/L 时表达减弱，10 mg/L以上表达逐渐增强。Caspase 7在各组的表达未见明显差异。Caspase 10表达随氟化钠剂量增高而减弱。NaF 处理 24 h 后发现成骨细胞内有 48 条 miRNA 表达上调，4条 miRNA表达下调；NaF处理 48 h有21条 miRNA 表达上调，2 条 miRNA 表达下调；两个时间点共同上调miRNA 有 9 条，共同下调miRNA有1条；在原代培养的成骨细胞建立的染氟模型中20 mg/L NaF处理12h和24h后发现成骨细胞内共有128条miRNA表达上调,36条miRNA表达下调；40 mg/L组12h和24h后有130条miRNA表达上调,29条miRNA表达下调；两个时间点共同上调miRNA有72条,共同下调miRNA有2条。Caspase 14在染氟小鼠模型骨组织中亦有表达，染氟组表达均高于对照组，且有逐渐增高趋势。人的标本中未能检出miRNA的表达