本文選題：鎘 + 大鼠��； 參考：《廣州醫(yī)學(xué)院》2012年碩士論文

【摘要】：1.背景 鎘(Cadmium，Cd)是一種廣泛存在于環(huán)境中的重金屬污染物和致癌物。由于其在體內(nèi)能長(zhǎng)期蓄積，不易排除，故易對(duì)機(jī)體產(chǎn)生慢性及遠(yuǎn)期毒效應(yīng)，甚至導(dǎo)致人類與哺乳動(dòng)物多種腫瘤如肺癌、腎癌、前列腺癌和胰腺癌等。1993年國(guó)際癌癥研究組織已將鎘列為第一類致癌物，但其分子毒作用機(jī)制仍不清楚。近年來，來自不同研究領(lǐng)域分別試圖從整體、器官、細(xì)胞和分子水平等方面來闡明鎘對(duì)機(jī)體的毒作用及其機(jī)制，特別是鎘低水平長(zhǎng)期毒作用已經(jīng)成為毒理學(xué)研究的熱點(diǎn)。有研究認(rèn)為，DNA修復(fù)功能被抑制可能是金屬毒作用的重要機(jī)制之一，但關(guān)于DNA修復(fù)基因在亞慢性鎘暴露動(dòng)物體內(nèi)的表達(dá)變化卻鮮見報(bào)道。因此本研究利用課題組前期已成功建立的大鼠亞慢性鎘暴露模型，對(duì)大鼠靶器官DNA修復(fù)基因XRCC1和hOGG1表達(dá)變化進(jìn)行研究，為鎘化物低水平長(zhǎng)期致機(jī)體害的分子毒作用機(jī)制提供新的實(shí)驗(yàn)數(shù)據(jù)。 2.方法 2.1根據(jù)XRCC1和hOGG1基因的mRNA序列，用Primer Express5.0軟件分別設(shè)計(jì)XRCC1和hOGG1基因的上下游引物。應(yīng)用Trizol試劑盒提取大鼠肝、腎、心與肺臟器中總RNA，經(jīng)逆轉(zhuǎn)錄后用實(shí)時(shí)熒光定量PCR(Taqman和SYBR Green)方法檢測(cè)XRCC1和hOGG1基因的表達(dá)情況。 2.2用總蛋白提取液并按照其說明提取大鼠肝、腎、心與肺等靶器官中總蛋白，經(jīng)蛋白定量后采用Western blot檢測(cè)方法分析XRCC1和hOGG1蛋白的表達(dá)改變。同時(shí)，分析大鼠肝、腎、心和肺臟器的XRCC1和hOGG1表達(dá)水平與相應(yīng)各臟器鎘蓄積量及其病理損害的關(guān)系。 2.3所有實(shí)驗(yàn)均重復(fù)三次，實(shí)驗(yàn)數(shù)據(jù)以x±s表示，如果原始資料為偏態(tài)分布則用對(duì)數(shù)(log)進(jìn)行轉(zhuǎn)換以達(dá)到正態(tài)化，運(yùn)用t檢驗(yàn)、單因素方差分析、Games-Howe11檢驗(yàn)和相關(guān)分析等對(duì)數(shù)據(jù)進(jìn)行處理，P0.05有統(tǒng)計(jì)學(xué)意義。 3.結(jié)果 3.1通過Taqman實(shí)時(shí)熒光定量PCR分析，在鎘高、中、低劑量染毒大鼠中，，各種鎘靶器官的XRCC1基因表達(dá)量以TBP為內(nèi)參照進(jìn)行標(biāo)化計(jì)算，發(fā)現(xiàn)XRCC1mRNA表達(dá)水平在肝臟分別是相應(yīng)對(duì)照組的44%，31%和13%，在腎臟分別是相應(yīng)對(duì)照組的62%，29%和15%，在心臟分別是相應(yīng)對(duì)照組的42%，30%和15%；在肺臟的分別是相應(yīng)對(duì)照組的47%，28%和19%，提示隨著大鼠CdC12暴露水平的升高而其肝、腎、心與肺等靶器官的XRCC1基因表達(dá)水平明顯下調(diào)，存在良好的劑量-反應(yīng)關(guān)系(P0.01)。 3.2經(jīng)過SYBR Green實(shí)時(shí)熒光定量PCR檢測(cè)，在鎘高、中、低劑量染毒大鼠中，各種鎘靶器官的hOGG1基因表達(dá)量以β-actin為內(nèi)參照進(jìn)行標(biāo)化計(jì)算，觀察到hOGG1mRNA表達(dá)水平在肝臟分別是相應(yīng)對(duì)照組的67%，50%和16%；在腎臟分別是相應(yīng)對(duì)照組的40%，21%和12%，在心臟分別是相應(yīng)對(duì)照組的72%，59%和27%；在肺臟的分別是相應(yīng)對(duì)照組的75%，52%和29%，顯示隨著大鼠CdC12暴露水平的升高而其肝、腎、心與肺臟器的hOGG1基因表達(dá)水平顯著下調(diào)，有明顯的劑量-反應(yīng)關(guān)系(P0.01)。 3.3運(yùn)用Western b1ot檢測(cè)與分析，可見大鼠肝、腎臟器的XRCC1和hOGG1蛋白質(zhì)在各染鎘大鼠組中均出現(xiàn)不同程度的表達(dá)下調(diào)，與相應(yīng)對(duì)照組相比有下降趨勢(shì)，此現(xiàn)象在肝組織中最為明顯，進(jìn)一步確證了XRCC1和hOGG1基因在鎘暴霸大鼠靶器官中的下調(diào)表達(dá)。同時(shí)通過相關(guān)分析也顯示大鼠肝、腎、心和肺臟器的XRCC1和hOGG1表達(dá)水平與相應(yīng)各臟器鎘蓄積量及其病理損害密切相關(guān)，提示DNA修復(fù)基因XRCC1和hOGG1的表達(dá)變化可考慮作鎘暴露及其效應(yīng)的生物標(biāo)志物。 4.結(jié)論 4.1長(zhǎng)期低水平的鎘化物暴露可導(dǎo)致大鼠肝、腎、心、肺等靶器官的XRCC1和hOGG1基因表達(dá)水平明顯下調(diào)，并與染鎘劑量存在良好的劑量-反應(yīng)關(guān)系，表明某些DNA修復(fù)基因如XRCC1和hOGG1的表達(dá)改變?cè)阪k分子毒作用機(jī)制中可能起著重要的作用。 4.2在亞慢性鎘中毒大鼠模型中發(fā)現(xiàn)其肝、腎、心和肺等主要內(nèi)部臟器的XRCC1和hOGG1表達(dá)水平與相應(yīng)各臟器鎘蓄積量及其病理損害密切相關(guān)，提示XRCC1和hOGG1基因的表達(dá)改變有可能成為一個(gè)有價(jià)值的鎘亞慢性暴露及其效應(yīng)的生物標(biāo)志物。
[Abstract]:1. background
Cadmium (Cd) is a kind of heavy metal contaminant and carcinogen that exists widely in the environment. Because it can accumulate in the body for a long time, it is not easy to eliminate, so it is easy to produce chronic and long-term toxic effects to the body, and even lead to human and mammal tumors such as lung cancer, kidney cancer, prostate cancer and pancreatic cancer,.1993 international cancer research organization already Cadmium is classified as the first carcinogen, but the mechanism of its molecular toxicity is still unclear. In recent years, different research fields have tried to elucidate the toxic effects and mechanisms of cadmium to the body from the whole, organ, cell and molecular levels, especially the low cadmium level long-term toxicity of cadmium, which has become a hot spot in toxicology research. The inhibition of DNA repair function may be one of the important mechanisms of metal toxicity, but the changes in the expression of DNA repair genes in subchronic cadmium exposed animals are rarely reported. Therefore, the subchronic cadmium exposure model in rats has been successfully established in the previous study group, and the expression of the DNA repair gene XRCC1 and hOGG1 in the target organs of the rat is changed. The study will provide new experimental data for the mechanism of low molecular weight toxicity of cadmium.
2. method
2.1 according to the mRNA sequence of XRCC1 and hOGG1 gene, the upper and lower primers of XRCC1 and hOGG1 genes were designed with Primer Express5.0 software respectively. The total RNA in rat liver, kidney, heart and lung were extracted with Trizol kit. The expression of the gene was detected by real time fluorescent quantitative PCR (Taqman and SYBR).
2.2 the total protein extracted from the rat liver, kidney, heart, lung and other target organs was extracted with the total protein extract. The expression changes of XRCC1 and hOGG1 protein were analyzed by Western blot assay after the protein quantitative analysis. Meanwhile, the expression of XRCC1 and hOGG1 in the liver, kidney, heart and lung organs of rats and the cadmium accumulation and pathology of the corresponding organs and their pathology were analyzed. The relationship of damage.
2.3 all the experiments were repeated three times. The experimental data were expressed as x + s. If the original data were partial distribution, the data were converted to normalization by logarithm (log). T test, single factor analysis of variance, Games-Howe11 test and correlation analysis were used to deal with the data, and P0.05 had statistical significance.
3. results
3.1 by Taqman real-time fluorescence quantitative PCR analysis, the XRCC1 gene expression of various cadmium target organs in cadmium high, middle and low dose rats was calculated with TBP as internal reference, and the expression level of XRCC1mRNA in the liver was 44%, 31% and 13% in the corresponding control group, respectively, 62%, 29% and 15% in the corresponding control group, respectively, in the heart. Don't be 42%, 30% and 15% of the corresponding control group; the lungs were 47%, 28% and 19% in the corresponding control group, suggesting that the level of XRCC1 gene expression in the target organs such as liver, kidney, heart and lung was obviously down, and there was a good dose response system (P0.01) with the increase of CdC12 exposure level in the rats.
3.2 after SYBR Green real-time fluorescence quantitative PCR detection, the hOGG1 gene expression of various cadmium target organs in cadmium high, middle and low dose rats was labeled with beta -actin as internal reference, and the expression level of hOGG1mRNA in the liver was 67%, 50% and 16% in the corresponding control group, respectively, and 40%, 21% and 12 in the corresponding control group respectively. The heart was 72%, 59% and 27% in the corresponding control group, and the lungs were 75%, 52% and 29% in the corresponding control group. The hOGG1 gene expression levels in the liver, kidney, heart and lung were significantly down down with the increase of CdC12 exposure level in the rats, and there was a significant dose reverse relationship (P0.01).
3.3 Western b1ot detection and analysis showed that the expression of XRCC1 and hOGG1 protein in rat liver and kidney organ decreased in varying degrees, and decreased in comparison with the corresponding control group. This phenomenon was the most obvious in the liver tissue, and further confirmed the XRCC1 and hOGG1 genes in the target organs of the rat of cadmium tyrants. The expression of XRCC1 and hOGG1 in rat liver, kidney, heart and lung organs was also closely related to the cadmium accumulation and pathological damage of various organs, suggesting that the expression of DNA repair gene XRCC1 and hOGG1 could be considered as a biomarker for cadmium exposure and its effect.
4. conclusion
4.1 long term low level cadmium exposure can lead to the obvious downregulation of XRCC1 and hOGG1 gene expression levels in target organs such as liver, kidney, heart, lung and other target organs, and there is a good dose response relationship with the dose of cadmium, suggesting that the expression of some DNA repair genes, such as XRCC1 and hOGG1, may play an important role in the mechanism of cadmium subtoxicity.
4.2 in the subchronic cadmium poisoning rat model, the expression of XRCC1 and hOGG1 in the main internal organs of the liver, kidney, heart and lung is closely related to the cadmium accumulation and pathological damage of the corresponding organs. It is suggested that the expression of XRCC1 and hOGG1 gene may be a valuable biomarker for cadmium subchronic exposure and its effect.
【學(xué)位授予單位】：廣州醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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相關(guān)期刊論文 前3條

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本文編號(hào)：1927092