【摘要】：重金屬鎘(Cadmium, Cd)是一種重要的工業(yè)和環(huán)境污染因素,在空氣、土壤和水中廣泛存在,被美國毒理管理委員會(ATSDR)列為第六位危及人體健康的有毒物質。Cd具有內分泌干擾作用,可以干擾生物體激素的合成、釋放、轉運、與受體結合、代謝等途徑,從而影響內分泌系統(tǒng)功能,破壞機體內環(huán)境的協(xié)調和穩(wěn)定。有資料顯示,Cd可誘導睪丸、附睪和精囊腺等組織器官發(fā)生結構和功能上的退行性變化,引起生精障礙,甚至不育。流行病學數(shù)據表明,Cd能夠增加個體罹患前列腺癌(Prostate Cancer, PCa)的風險。前列腺等生殖相關組織器官都是內分泌依賴的器官,其生長、分化受到雄激素水平的調節(jié),而雄激素發(fā)揮作用依賴于其受體AR (Androgen receptor)。AR是一個轉錄因子,具有轉錄激活活性,可激活下游基因的轉錄,同時AR信號通路在前列腺的生長發(fā)育、疾病發(fā)生中發(fā)揮著極其重要的作用。目前關于Cd對AR介導的干擾作用不甚清楚,其機制尚未明確。 本課題以雄激素受體AR為切入點研究鎘的內分泌干擾毒性,探討Cd對AR表達、轉錄活性及下游信號通路的影響,并對其影響機制進行研究,以此來尋找Cd為代表的重金屬類內分泌干擾物的毒理學機制,為其他物質的雄性激素內分泌干擾毒性篩選提供新的方法,進一步為鎘污染所造成疾病的預防控制提供新的策略。 首先利用CCK8實驗研究了Cd對LNCaP細胞的細胞毒性作用,發(fā)現(xiàn)16μ.M以下濃度無明顯細胞毒性；流式細胞技術檢測不同濃度的Cd處理對LNCaP細胞周期的影響,發(fā)現(xiàn)Cd可以使LNCaP細胞的S期增加,這表明Cd可能促進細胞的增殖；利用熒光素酶試驗,將帶有熒光素酶報告基因的雄激素反應元件(Androgen response element, ARE)和AR共轉染293T細胞,經不同濃度Cd處理,分析Cd對AR轉錄活性的影響,發(fā)現(xiàn)Cd可增強AR的轉錄活性；然后利用qRT-PCR和western檢測不同濃度Cd處理后AR及其調控的下游靶基因——前列腺特異性抗原(Prostate specific antigen, PSA) mRNA和蛋白表達水平變化,發(fā)現(xiàn)AR可增加PSA的表達,但AR本身的表達量沒有明顯改變,這表明Cd不是通過改變AR的表達水平影響AR的功能。 類泛素化修飾(SUMO化)是一種重要的翻譯后修飾,AR經類泛素化修飾后轉錄活性降低而表達量不變。文獻報道,去類泛素化酶SENP1可裂解類泛素與AR之間的共價連接,降低AR的類泛素化水平。因此我們推測Cd很可能通過影響SENP1調節(jié)AR的轉錄活性。我們檢測了Cd對可破壞AR SUMO化的蛋白酶1(SUMO specific protease, SENP1) mRNA和蛋白表達水平的變化,發(fā)現(xiàn)Cd增加SENP1的表達；繼而利用基因沉默技術,檢測LNCaP細胞中的SENP1沉默后,AR及其下游靶基因PSA的表達,發(fā)現(xiàn)通過SENP1調控AR的活性；進一步檢測了Cd對/AR SUMO化水平的影響,發(fā)現(xiàn)Cd處理后AR分子的SUMO化水平降低。綜上所述,Cd通過SENP1降低雄激素受體AR的SUMO化調節(jié)AR的活性。以上結果分析了鎘雄激素內分泌干擾毒性的一種新機制,為其他物質的內分泌毒性篩選提供新的方法,也為尋找環(huán)境Cd污染所致疾病的預防控制的新策略提供理論支持。
[Abstract]:Cadmium (Cd) is an important industrial and environmental pollution factor, which exists widely in air, soil and water. The American toxicology Management Committee (ATSDR) has been listed as sixth toxic substances that endanger human health,.Cd has endocrine disrupting effects, which can interfere with the synthesis, release, transport, and metabolism of the hormones. It has been shown that Cd can induce degenerative changes in the structure and function of the tissues and organs such as testis, epididymis and seminal vesicles, causing spermatogenesis and even infertility. Epidemiological data show that Cd can increase the prostate cancer of the individual (Prostate Cancer, PCa) risk. Prostate and other reproductive organs and organs are endocrine dependent organs, and their growth and differentiation are regulated by androgen levels, while androgens play a role depending on their receptor AR (Androgen receptor).AR as a transcription factor, which has a transcriptional activation activity that activates the transcription of the downstream genes, while AR signals are used. The pathway plays an important role in the growth and development of the prostate and the occurrence of the disease. At present, the interference of Cd on AR is not clear, and its mechanism is not clear.
In this study, the endocrine disrupting toxicity of cadmium was studied with androgen receptor AR as a breakthrough point. The effects of Cd on AR expression, transcriptional activity and downstream signal pathway were investigated, and the mechanism of its influence was studied in order to find the toxicological mechanism of the endocrine disruptors, represented by Cd, and to interfere with the male hormone endocrine disrupting drugs of other substances. Sex screening provides new ways to further provide new strategies for disease prevention and control caused by cadmium pollution.
First, the cytotoxic effect of Cd on LNCaP cells was studied by CCK8 experiment. It was found that there was no obvious cytotoxicity of the concentration below 16.M. Flow cytometry was used to detect the effect of Cd on the cycle of LNCaP cells at different concentrations. It was found that Cd could increase the S phase of LNCaP cells, which indicates that Cd may promote cell proliferation; luciferase test is used. The Androgen response element (ARE) and AR were co transfected to 293T cells, and the effect of Cd on AR transcriptional activity was analyzed by Cd treatment at different concentrations. It was found that Cd enhanced the AR transcriptional activity. The gene, Prostate specific antigen (PSA) mRNA and protein expression level changes, found that AR can increase the expression of PSA, but the expression of AR itself is not significantly changed, which indicates that Cd does not affect AR by changing the expression level of AR.
Ubiquitination modification (SUMO) is an important post-translational modification, and the expression of AR after ubiquitin modification is reduced and the amount of expression is unchanged. It is reported that de ubiquitinase SENP1 can cleave the covalent connection between AR and reduce the ubiquitination level of AR. Therefore, we speculate that Cd is likely to regulate the transcription of AR by affecting SENP1. We detected the changes in the expression level of protease 1 (SUMO specific protease, SENP1) mRNA and protein that could destroy AR SUMO, and found that Cd increased the expression of SENP1. Then, the expression of SENP1 silencing in LNCaP cells was detected by gene silencing, and the expression of the target gene and its downstream target gene were detected. The effect of Cd on the level of /AR SUMO was further detected. It was found that the SUMO level of AR molecules decreased after Cd treatment. To sum up, Cd regulates AR activity by SUMO conversion of androgen receptor AR through SENP1, and the above results are a new mechanism for the endocrine disrupting toxicity of cadmium androgen, which provides a new method for screening the endocrine toxicity of other substances. The method also provides theoretical support for finding new strategies for prevention and control of diseases caused by environmental pollution caused by Cd.
【學位授予單位】：蘭州大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：R114

【共引文獻】

相關期刊論文 前1條

1 李裕;張強;王潤元;肖國舉;王勝;;鎘的致癌性與食品中鎘的生物有效性[J];生命科學;2010年02期

相關會議論文 前1條

1 李裕;張強;王潤元;肖國舉;;食品中鎘的生物有效性與影響因素[A];第27屆中國氣象學會年會干旱半干旱區(qū)地氣相互作用分會場論文集[C];2010年

，

本文編號：2128717