氟对体外培养的成骨细胞中Ras基因表达的影响
发布时间：2018-10-31 21:19
【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%qL、2.5mg/L、5mg%qL、10mg/L和20mg%qL;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%qL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%qL染氟组的成骨细胞Ras基因则为弱阳性表达;2.5mg%qL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%qL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%qL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。
[Abstract]:Objective: to investigate the effects of different doses of fluoride on the expression of Ras gene in mouse osteoblasts from the level of protein and mRNA expression on the basis of verifying and improving the common methods of primary culture of mouse osteoblasts. At the same time, the effects of different doses of fluoride on the expression of RasmRNA in human osteoblasts were detected. Methods: the primary culture method of mouse osteoblasts was established and identified by alkaline phosphatase (ALP) and calcium nodule staining. The fluoride doses were: 0 mg / L 2.5 mg / L ~ 5 mg / L ~ 10 mg / L and 20 mg / L ~ 20 mg / L respectively. The expression of Ras protein and mRNA in the osteoblasts of mice exposed to fluoride were detected by immunohistochemistry, in situ hybridization, immunological imprinting and RT-PCR (reverse transcri ption polymerase chain reaction). The expression of RasmRNA in human osteoblasts was determined by Real Time RT-PCR. Results: the method of cell culture was reproducible, the osteoblast was stable, the purification effect was good, and the model of fluorosis in vitro was established. After 10 days of fluorosis in mouse osteoblasts, the results of immunohistochemistry and in situ hybridization showed that there was a strong positive expression of Ras gene in osteoblasts of control group, 2.5mg%qL group and 5.0mg/L group, but 10.0 mg 路L ~ (-1) 路L ~ (-1) of Ras gene in osteoblasts. The expression of Ras gene in osteoblasts of 20.0mg%qL group was weakly positive. Compared with the control group, the positive intensity of 2.5mg/L group was higher than that of the control group, and the range of expression was significantly larger than that of the control group. Compared with the control group, the positive intensity of the other fluorine groups was lower than that of the control group, and the range of expression was obviously smaller than that of the control group. The number of positive cells in each fluorine group was significantly different from that in the control group (P < 0. 05). The number of positive cells in the 2.5mg/L group was higher than that in the control group, and that in the other fluorinated groups was lower than that in the control group. The number of positive cells in 2.5mg/L and 5.0mg%qL groups was higher than that in other fluoride groups (P < 0. 05), and the number of positive cells in 2.5mg/L group was the highest (P < 0. 05). The relative quantitative results of RasmRNA in osteoblasts of mice showed that the 2.5mg/L group was higher than the control group, and the other groups were less than the control group, and the 2.5mg/L group was higher than the other fluorine group. The relative quantification of RasmRNA in fluoride group decreased gradually with the increase of dose. The relative quantitative results of Ras protein in mouse osteoblasts showed that the relative quantification of 2.5mg/L and 5.0mg/L was higher than that of the control group, and that of the other fluorine-exposed groups was smaller than that of the control group. At the same time, the relative quantification of fluorine histone in 5mg%qL was higher than that in other fluorine groups. The relative quantitative analysis of RasmRNA in human osteoblasts showed that the relative quantity of RasmRNA in human osteoblasts treated with 2.5mg/L and above doses of fluoride was lower than that in the control group. Conclusion: fluoride can affect the expression of Ras gene protein and mRNA in mouse osteoblasts after the action of fluoride, which shows that low dose fluoride can promote the expression of Ras gene protein and mRNA. High dose fluoride inhibited the expression of Ras gene protein and mRNA. The expression of RasmRNA in human osteoblasts was affected by fluoride, and the expression of RasmRNA decreased with the increase of dose.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
本文编号：2303587
[Abstract]:Objective: to investigate the effects of different doses of fluoride on the expression of Ras gene in mouse osteoblasts from the level of protein and mRNA expression on the basis of verifying and improving the common methods of primary culture of mouse osteoblasts. At the same time, the effects of different doses of fluoride on the expression of RasmRNA in human osteoblasts were detected. Methods: the primary culture method of mouse osteoblasts was established and identified by alkaline phosphatase (ALP) and calcium nodule staining. The fluoride doses were: 0 mg / L 2.5 mg / L ~ 5 mg / L ~ 10 mg / L and 20 mg / L ~ 20 mg / L respectively. The expression of Ras protein and mRNA in the osteoblasts of mice exposed to fluoride were detected by immunohistochemistry, in situ hybridization, immunological imprinting and RT-PCR (reverse transcri ption polymerase chain reaction). The expression of RasmRNA in human osteoblasts was determined by Real Time RT-PCR. Results: the method of cell culture was reproducible, the osteoblast was stable, the purification effect was good, and the model of fluorosis in vitro was established. After 10 days of fluorosis in mouse osteoblasts, the results of immunohistochemistry and in situ hybridization showed that there was a strong positive expression of Ras gene in osteoblasts of control group, 2.5mg%qL group and 5.0mg/L group, but 10.0 mg 路L ~ (-1) 路L ~ (-1) of Ras gene in osteoblasts. The expression of Ras gene in osteoblasts of 20.0mg%qL group was weakly positive. Compared with the control group, the positive intensity of 2.5mg/L group was higher than that of the control group, and the range of expression was significantly larger than that of the control group. Compared with the control group, the positive intensity of the other fluorine groups was lower than that of the control group, and the range of expression was obviously smaller than that of the control group. The number of positive cells in each fluorine group was significantly different from that in the control group (P < 0. 05). The number of positive cells in the 2.5mg/L group was higher than that in the control group, and that in the other fluorinated groups was lower than that in the control group. The number of positive cells in 2.5mg/L and 5.0mg%qL groups was higher than that in other fluoride groups (P < 0. 05), and the number of positive cells in 2.5mg/L group was the highest (P < 0. 05). The relative quantitative results of RasmRNA in osteoblasts of mice showed that the 2.5mg/L group was higher than the control group, and the other groups were less than the control group, and the 2.5mg/L group was higher than the other fluorine group. The relative quantification of RasmRNA in fluoride group decreased gradually with the increase of dose. The relative quantitative results of Ras protein in mouse osteoblasts showed that the relative quantification of 2.5mg/L and 5.0mg/L was higher than that of the control group, and that of the other fluorine-exposed groups was smaller than that of the control group. At the same time, the relative quantification of fluorine histone in 5mg%qL was higher than that in other fluorine groups. The relative quantitative analysis of RasmRNA in human osteoblasts showed that the relative quantity of RasmRNA in human osteoblasts treated with 2.5mg/L and above doses of fluoride was lower than that in the control group. Conclusion: fluoride can affect the expression of Ras gene protein and mRNA in mouse osteoblasts after the action of fluoride, which shows that low dose fluoride can promote the expression of Ras gene protein and mRNA. High dose fluoride inhibited the expression of Ras gene protein and mRNA. The expression of RasmRNA in human osteoblasts was affected by fluoride, and the expression of RasmRNA decreased with the increase of dose.
【学位授予单位】：新疆医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R363
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