氟對(duì)體外培養(yǎng)的成骨細(xì)胞中Ras基因表達(dá)的影響
發(fā)布時(shí)間：2018-10-31 21:19
【摘要】： 目的:在驗(yàn)證及改良小鼠成骨細(xì)胞原代培養(yǎng)常用方法的基礎(chǔ)上,分別從蛋白質(zhì)及其mRNA表達(dá)水平檢測(cè)不同劑量氟對(duì)小鼠成骨細(xì)胞中Ras基因的影響;同時(shí)檢測(cè)不同劑量氟對(duì)人成骨細(xì)胞中RasmRNA表達(dá)量的影響。方法:建立與改良小鼠成骨細(xì)胞的原代培養(yǎng)方法,通過(guò)堿性磷酸酶和鈣結(jié)節(jié)染色方法進(jìn)行細(xì)胞來(lái)源鑒定;染氟劑量分別為:0mg%qL、2.5mg/L、5mg%qL、10mg/L和20mg%qL;利用免疫組化、原位雜交、免疫印記和RT-PCR(reverse transcri ption polymerase chain reaction)的方法檢測(cè)染氟后小鼠成骨細(xì)胞中Ras蛋白及mRNA表達(dá)水平;采用Real Time RT-PCR的方法測(cè)定染氟后人成骨細(xì)胞內(nèi)RasmRNA表達(dá)量的變化。結(jié)果:改良后的細(xì)胞培養(yǎng)方法重復(fù)性好、成骨細(xì)胞生長(zhǎng)穩(wěn)定、純化效果好,完全適和建立氟中毒的體外模型。小鼠成骨細(xì)胞染氟10天后,免疫組化和原位雜交結(jié)果顯示:對(duì)照組、2.5mg%qL染氟組及5.0mg/L染氟組成骨細(xì)胞中Ras基因均有較強(qiáng)陽(yáng)性表達(dá),但10.0mg/L、20.0mg%qL染氟組的成骨細(xì)胞Ras基因則為弱陽(yáng)性表達(dá);2.5mg%qL組與對(duì)照組比較,2.5mg/L組的陽(yáng)性強(qiáng)度高于對(duì)照組,表達(dá)范圍也明顯大于對(duì)照組;而其余各染氟組與對(duì)照組比較,陽(yáng)性強(qiáng)度低于對(duì)照組,表達(dá)范圍也明顯小于對(duì)照組。陽(yáng)性細(xì)胞計(jì)數(shù)結(jié)果顯示:各染氟組陽(yáng)性表達(dá)細(xì)胞數(shù)與對(duì)照組比較有統(tǒng)計(jì)學(xué)差異(P＜0.05),2.5mg/L染氟組高于對(duì)照組,其它染氟組低于對(duì)照組;2.5mg/L和5.0mg%qL染氟組陽(yáng)性細(xì)胞數(shù)高于其它染氟組(P＜0.05),2.5mg/L組陽(yáng)性細(xì)胞數(shù)最高。小鼠成骨細(xì)胞的RasmRNA相對(duì)定量結(jié)果顯示:2.5mg/L染氟組高于對(duì)照組,其它染氟組均少于對(duì)照組;同時(shí)2.5mg/L染氟組大于其它染氟組;染氟組的RasmRNA相對(duì)定量有隨著劑量增加而逐漸降低的趨勢(shì)。小鼠成骨細(xì)胞的Ras蛋白相對(duì)定量結(jié)果顯示:2.5mg/L與5.0mg/L染氟組蛋白相對(duì)定量大于對(duì)照組,其它染氟組均小于對(duì)照組;同時(shí)5mg%qL染氟組蛋白相對(duì)定量大于其它染氟組。人成骨細(xì)胞中RasmRNA相對(duì)定量顯示:2.5mg/L及以上劑量氟作用下人的成骨細(xì)胞中RasmRNA相對(duì)定量小于對(duì)照組。結(jié)論:氟作用于小鼠成骨細(xì)胞后可以影響小鼠成骨細(xì)胞的Ras基因蛋白及mRNA的表達(dá),表現(xiàn)為雙向作用即低劑量的氟對(duì)Ras基因蛋白及mRNA表達(dá)有促進(jìn)作用,高劑量的氟抑制Ras基因蛋白及mRNA表達(dá)。氟作用于人成骨細(xì)胞后可以影響人成骨細(xì)胞的RasmRNA的表達(dá),本實(shí)驗(yàn)劑量中表現(xiàn)為RasmRNA表達(dá)量隨著劑量增加而減少。
[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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
本文編號(hào)：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.
【學(xué)位授予單位】：新疆醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R363
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 劉鳳,劉開(kāi)泰,李靈芝,張永亮,姚華;氟化物對(duì)成骨細(xì)胞增殖和分化的影響[J];地方病通報(bào);2004年03期
2 邵紅,劉開(kāi)泰,姚華,張躍新,劉文亞,冀芳,張婕;氟中毒患者外周血淋巴細(xì)胞基因表達(dá)水平的研究[J];地方病通報(bào);2005年02期
3 劉東生 ,陳慶沐 ,余志成 ,袁芷云;我國(guó)地方性氟病的地球化學(xué)問(wèn)題[J];地球化學(xué);1980年01期
4 王長(zhǎng)松,陳燕平,劉家騮,于燕妮,唐俊杰;氟在體外對(duì)兔成骨細(xì)胞增殖行為的影響[J];中國(guó)地方病防治雜志;2004年01期
5 陳慶沐,劉玉蘭;氟的土壤地球化學(xué)與地方性氟中毒[J];環(huán)境科學(xué);1981年06期
6 劉開(kāi)泰,馬麗英,李玲芝,張永亮,王國(guó)荃;氟對(duì)體外培養(yǎng)長(zhǎng)骨生長(zhǎng)發(fā)育、形態(tài)結(jié)構(gòu)和生化指標(biāo)的影響[J];環(huán)境與健康雜志;2003年04期
7 張偉國(guó),王麗珍,劉正;氟對(duì)鼠顱骨成骨細(xì)胞基質(zhì)蛋白表達(dá)的影響[J];上海口腔醫(yī)學(xué);1998年02期
8 邊建朝,葉平,咸樹(shù)梅,紀(jì)慶憲,劉源,管淑芬,劉永平;慢性氟中毒大鼠體氟積累排泄動(dòng)態(tài)變化及硒的影響[J];衛(wèi)生研究;1997年04期
9 孫殿軍,李忠之,張政;21世紀(jì)中國(guó)地方病防治主要工作研討[J];中國(guó)地方病學(xué)雜志;2000年06期
10 殷大奎;殷大奎副部長(zhǎng)在全國(guó)防治地方性氟中毒(砷中毒)改水工作經(jīng)驗(yàn)交流會(huì)上的講話[J];中國(guó)地方病學(xué)雜志;2001年04期
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