DNA修复相关基因在镉亚慢性暴露大鼠中的表达改变

发布时间：2018-05-24 01:05

本文选题：镉 + 大鼠　；参考：《广州医学院》2012年硕士论文

【摘要】：1.背景镉(Cadmium，Cd)是一种广泛存在于环境中的重金属污染物和致癌物。由于其在体内能长期蓄积，不易排除，故易对机体产生慢性及远期毒效应，甚至导致人类与哺乳动物多种肿瘤如肺癌、肾癌、前列腺癌和胰腺癌等。1993年国际癌症研究组织已将镉列为第一类致癌物，但其分子毒作用机制仍不清楚。近年来，来自不同研究领域分别试图从整体、器官、细胞和分子水平等方面来阐明镉对机体的毒作用及其机制，特别是镉低水平长期毒作用已经成为毒理学研究的热点。有研究认为，DNA修复功能被抑制可能是金属毒作用的重要机制之一，但关于DNA修复基因在亚慢性镉暴露动物体内的表达变化却鲜见报道。因此本研究利用课题组前期已成功建立的大鼠亚慢性镉暴露模型，对大鼠靶器官DNA修复基因XRCC1和hOGG1表达变化进行研究，为镉化物低水平长期致机体害的分子毒作用机制提供新的实验数据。 2.方法 2.1根据XRCC1和hOGG1基因的mRNA序列，用Primer Express5.0软件分别设计XRCC1和hOGG1基因的上下游引物。应用Trizol试剂盒提取大鼠肝、肾、心与肺脏器中总RNA，经逆转录后用实时荧光定量PCR(Taqman和SYBR Green)方法检测XRCC1和hOGG1基因的表达情况。 2.2用总蛋白提取液并按照其说明提取大鼠肝、肾、心与肺等靶器官中总蛋白，经蛋白定量后采用Western blot检测方法分析XRCC1和hOGG1蛋白的表达改变。同时，分析大鼠肝、肾、心和肺脏器的XRCC1和hOGG1表达水平与相应各脏器镉蓄积量及其病理损害的关系。 2.3所有实验均重复三次，实验数据以x±s表示，如果原始资料为偏态分布则用对数(log)进行转换以达到正态化，运用t检验、单因素方差分析、Games-Howe11检验和相关分析等对数据进行处理，P0.05有统计学意义。 3.结果 3.1通过Taqman实时荧光定量PCR分析，在镉高、中、低剂量染毒大鼠中，，各种镉靶器官的XRCC1基因表达量以TBP为内参照进行标化计算，发现XRCC1mRNA表达水平在肝脏分别是相应对照组的44%，31%和13%，在肾脏分别是相应对照组的62%，29%和15%，在心脏分别是相应对照组的42%，30%和15%；在肺脏的分别是相应对照组的47%，28%和19%，提示随着大鼠CdC12暴露水平的升高而其肝、肾、心与肺等靶器官的XRCC1基因表达水平明显下调，存在良好的剂量-反应关系(P0.01)。 3.2经过SYBR Green实时荧光定量PCR检测，在镉高、中、低剂量染毒大鼠中，各种镉靶器官的hOGG1基因表达量以β-actin为内参照进行标化计算，观察到hOGG1mRNA表达水平在肝脏分别是相应对照组的67%，50%和16%；在肾脏分别是相应对照组的40%，21%和12%，在心脏分别是相应对照组的72%，59%和27%；在肺脏的分别是相应对照组的75%，52%和29%，显示随着大鼠CdC12暴露水平的升高而其肝、肾、心与肺脏器的hOGG1基因表达水平显著下调，有明显的剂量-反应关系(P0.01)。 3.3运用Western b1ot检测与分析，可见大鼠肝、肾脏器的XRCC1和hOGG1蛋白质在各染镉大鼠组中均出现不同程度的表达下调，与相应对照组相比有下降趋势，此现象在肝组织中最为明显，进一步确证了XRCC1和hOGG1基因在镉暴霸大鼠靶器官中的下调表达。同时通过相关分析也显示大鼠肝、肾、心和肺脏器的XRCC1和hOGG1表达水平与相应各脏器镉蓄积量及其病理损害密切相关，提示DNA修复基因XRCC1和hOGG1的表达变化可考虑作镉暴露及其效应的生物标志物。 4.结论 4.1长期低水平的镉化物暴露可导致大鼠肝、肾、心、肺等靶器官的XRCC1和hOGG1基因表达水平明显下调，并与染镉剂量存在良好的剂量-反应关系，表明某些DNA修复基因如XRCC1和hOGG1的表达改变在镉分子毒作用机制中可能起着重要的作用。 4.2在亚慢性镉中毒大鼠模型中发现其肝、肾、心和肺等主要内部脏器的XRCC1和hOGG1表达水平与相应各脏器镉蓄积量及其病理损害密切相关，提示XRCC1和hOGG1基因的表达改变有可能成为一个有价值的镉亚慢性暴露及其效应的生物标志物。
[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.
【学位授予单位】：广州医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R114

【参考文献】