【作者】 钟近洁；

【导师】 刘开泰； 姚华； 杨晓燕；

【作者基本信息】 新疆医科大学 ， 环境卫生学， 2006， 博士

【摘要】 目的：我国是一个地方性氟中毒病情严重的国家，病的类型多，分布广，病情重，受威胁人口多。随着相关研究工作的深入，如果能够从基础医学多学科的角度，利用新的先进技术手段，探索对地方性氟中毒(地氟病)发病机制的认识，尤其是从分子水平阐明氟中毒发生、发展的机制，将为地氟病的防治提供新的科学理论或思路。在前期课题的基础上，我们选择了氟骨症相关基因MCM3(minichromosome maintenance deficient 3(S．cerevisiae))进行分子机制的实验研究。MCM3是一种与细胞DNA复制密切相关的基因，其在细胞中含量的多少以及在细胞内分布位置的不同可以反映细胞的生长状态。本课题在验证及改良小鼠成骨细胞原代培养常用方法的基础上，测定了成骨细胞处于不同浓度氟环境、不同作用时间时氟离子在细胞内的分布情况和浓度变化趋势；观察不同剂量氟作用于体外培养小鼠成骨细胞后其增殖情况；检测氟对成骨细胞中MCM3蛋白表达水平的影响及mRNA分布情况；同时检测氟对成骨细胞中MCM3mRNA量的影响。 方法：(1)建立与改良小鼠成骨细胞的原代培养方法，并通过碱性磷酸酶和钙结节染色方法进行细胞来源鉴定。(2)建立核磁共振波谱法检测成骨细胞内氟离子的方法，观察染氟后成骨细胞中氟离子在细胞内的分布趋势。(3)染氟组按照剂量分为5组，即0、5、10、20、40mg／L组，采用电镜、绘制生长曲线、MTT法、流式细胞仪的方法观察成骨细胞的增殖情况。(4)免疫组化、原位杂交的方法检测染氟后成骨细胞中MCM3蛋白质表达和mRNA的分布。(5)采用Real Time RT-PCR的方法测定染氟后小鼠和人成骨细胞内MCM3mRNA量的变化。更多还原

【Abstract】 Objective: Fluorosis still is very severe in our country, it has many types and spreads widely, numbers of people who face to it are great. With more related research work being done, to explore the mole-mechanism of fluorosis with new techniques and skills of preclinical medical science can provide new path for the prevention of fluorosis. Based on the previous project, we choose MCM3 (minichromosome maintenance deficient 3) which is one of related genes of skeletal fluorosis to do further study. MCM3 is a kind of gene which has close relationship with DNA duplication, it’s quantity and distribution in cells reflect cell’s growth. After primary culture osteoblasts in mice being identified and improved, our project checks the concentration and distribution of fluoride ions in osteoblasts when osteoblasts are exposed different doses of fluoride with different period; observes the proliferation of osteoblasts exposed to fluoride in mice; detects MCM3 protein expression level, distribution and quantity of MCM3 mRNA of osteoblasts exposed to fluoride.Methods: (1) We built and improved the method of osteoblasts primary culture in vitro in mice, and identified the origination of osteoblasts by AKP and calcium nodes dyeing. (2) We observed the fluoride ions concentration and distribution in osteoblasts exposed to fluoride in mice by Nuclear Magenetic Resonance(NMR). (3)There were 5 experimental groups according to doses: 0, 5, 10, 20, 40mg/L. We observed the更多还原