【作者】 刘岳强；

【导师】 刘开泰； 杨晓燕； 刘继文；

【作者基本信息】 新疆医科大学 ， 劳动卫生与环境卫生学， 2007， 硕士

【摘要】 目的:在验证及改良小鼠成骨细胞原代培养常用方法的基础上,分别从蛋白质及其mRNA表达水平检测不同剂量氟对小鼠成骨细胞中Ras基因的影响;同时检测不同剂量氟对人成骨细胞中RasmRNA表达量的影响。方法:建立与改良小鼠成骨细胞的原代培养方法,通过碱性磷酸酶和钙结节染色方法进行细胞来源鉴定;染氟剂量分别为:0mg╱L、2.5mg/L、5mg╱L、10mg/L和20mg╱L;利用免疫组化、原位杂交、免疫印记和RT-PCR(reverse transcri ption polymerase chain reaction)的方法检测染氟后小鼠成骨细胞中Ras蛋白及mRNA表达水平;采用Real Time RT-PCR的方法测定染氟后人成骨细胞内RasmRNA表达量的变化。结果:改良后的细胞培养方法重复性好、成骨细胞生长稳定、纯化效果好,完全适和建立氟中毒的体外模型。小鼠成骨细胞染氟10天后,免疫组化和原位杂交结果显示:对照组、2.5mg╱L染氟组及5.0mg/L染氟组成骨细胞中Ras基因均有较强阳性表达,但10.0mg/L、20.0mg╱L染氟组的成骨细胞Ras基因则为弱阳性表达;2.5mg╱L组与对照组比较,2.5mg/L组的阳性强度高于对照组,表达范围也明显大于对照组;而其余各染氟组与对照组比较,阳性强度低于对照组,表达范围也明显小于对照组。阳性细胞计数结果显示:各染氟组阳性表达细胞数与对照组比较有统计学差异(P＜0.05),2.5mg/L染氟组高于对照组,其它染氟组低于对照组;2.5mg/L和5.0mg╱L染氟组阳性细胞数高于其它染氟组(P＜0.05),2.5mg/L组阳性细胞数最高。小鼠成骨细胞的RasmRNA相对定量结果显示:2.5mg/L染氟组高于对照组,其它染氟组均少于对照组;同时2.5mg/L染氟组大于其它染氟组;染氟组的RasmRNA相对定量有随着剂量增加而逐渐降低的趋势。小鼠成骨细胞的Ras蛋白相对定量结果显示:2.5mg/L与5.0mg/L染氟组蛋白相对定量大于对照组,其它染氟组均小于对照组;同时5mg╱L染氟组蛋白相对定量大于其它染氟组。人成骨细胞中RasmRNA相对定量显示:2.5mg/L及以上剂量氟作用下人的成骨细胞中RasmRNA相对定量小于对照组。结论:氟作用于小鼠成骨细胞后可以影响小鼠成骨细胞的Ras基因蛋白及mRNA的表达,表现为双向作用即低剂量的氟对Ras基因蛋白及mRNA表达有促进作用,高剂量的氟抑制Ras基因蛋白及mRNA表达。氟作用于人成骨细胞后可以影响人成骨细胞的RasmRNA的表达,本实验剂量中表现为RasmRNA表达量随着剂量增加而减少。更多还原

【Abstract】 Objective: To detect protein and RNA expression of Ras of mice osteoblasts and quantity of RasmRNA of human osteoblasts when osteoblasts was exposed different does of fluoride. Methods: After built and improved the method of osteobalsts primary culture in vitro in mice, we identified the origination of osteobalsts by AKP and calcium nodes dyeing; we detected Ras protein expression level and quantity of RasmRNA of Ras of mice osteoblasts which were exposed to different does of fluoride by immunohistoche-mistryl, hybridization in situ,western blottingting; to detect RasmRNA of human osteoblastsan which were exposed to different does of fluoride by Real Time PCR. Results: The culture method of osteobalsts in mice was repeated well. Cells grew stably and were purified successful which was basement of fluorosis model in vitro. All groups had Ras protein and RNA postitive expression after exposed to fluoride, but t postitive degree and scope in 2.5mg/L fluoride group were stonger and bigger than controal group and other fluoride groups of postitive degree and area were weaker and smaller than control group. Calculation of postitive osteobalsts numbers showed that it had statistic difference between the fluoride group and the control group. The numbers of 2.5mg/L fluoride groups were higher than the control group (p<0.05) and the numbers of the rest fluoride groups were lower than the control one (p<0.05). Among of these, numbers of 2.5mg/L fluoride groups were highest. The correspondence quantitation of RasmRNA in mice showed that the quantity of 2.5mg/L fluoride groups were more than the control group and the numbers of the rest fluoride groups were less than the control one, but the fluoride groups had decreased tendency with increase the the fluoride dose. The correspondence quantitation result of protein of Ras of mice showed that quantity of 2.5mg/Land 5.0mg/L fluoride groups were more than the control group and the numbers of the rest fluoride groups were less than the control one. The quantitation of RasmRNA of the fluoride groups were less the control one. Conclusions: Fluoride influences the Ras protein and mRNA expression of Ras in mice osteoblasts, which shows double action:low does of fluoride improves and high does of fluoride inhibits. Fluoride influences RasmRNA of human osteoblasts, and and the quantity of RasmRNA of human osteoblasts decrease with the increase of fluoride in the experience.更多还原