本文選題：芥子氣 + DNA加合物��； 參考：《中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院》2014年博士論文

【摘要】：芥子氣，英文全稱sulfur mustard（以下簡(jiǎn)稱SM，美軍代號(hào)HD），化學(xué)式為C4H8Cl2S，是雙功能烷基化試劑，也是糜爛性化學(xué)戰(zhàn)劑中最為重要的一種。芥子氣制備過(guò)程簡(jiǎn)單，毒性大且持續(xù)時(shí)間較長(zhǎng)，在戰(zhàn)爭(zhēng)中被數(shù)次使用，至今仍然威脅著世界和平和人民生命安全，有著“毒劑之王”之稱。然而，芥子氣的化學(xué)性質(zhì)決定了其毒性具有廣泛性，目前國(guó)際上關(guān)于其針對(duì)性解毒藥物的研究進(jìn)展緩慢，臨床救治也多將其視作燒傷處理。因此，深入研究芥子氣的毒理作用機(jī)制、篩選和開發(fā)其對(duì)抗特效治療藥物和臨床醫(yī)學(xué)干預(yù)方案具有重要現(xiàn)實(shí)意義，而這些亟需建立可靠的芥子氣溯源性檢測(cè)和毒理學(xué)評(píng)價(jià)方法。 長(zhǎng)期以來(lái)，國(guó)內(nèi)外學(xué)者對(duì)芥子氣毒理作用機(jī)制的研究不斷深入，并提出了一系列理論。雖然目前仍未得出確切結(jié)論，但是各國(guó)學(xué)者普遍認(rèn)同芥子氣對(duì)DNA分子的烷基化損傷是其毒理作用的始發(fā)機(jī)制以及細(xì)胞毒性與生物毒性的物質(zhì)基礎(chǔ)。因此DNA烷基化損傷及修復(fù)過(guò)程中產(chǎn)生的內(nèi)源性與外源性標(biāo)志物可視為芥子氣暴露后生物體損傷及恢復(fù)情況的有效評(píng)價(jià)指標(biāo)。 論文采用新興的分析毒理學(xué)相關(guān)方法，通過(guò)檢測(cè)芥子氣與DNA的烷基化加合物（以下簡(jiǎn)稱SM-DNA加合物）在體內(nèi)的時(shí)效與量效變化趨勢(shì)，實(shí)現(xiàn)對(duì)DNA的受損及修復(fù)過(guò)程的實(shí)時(shí)監(jiān)控，從而定量闡述芥子氣細(xì)胞毒性的物質(zhì)基礎(chǔ)與其毒性之間的相關(guān)性�，F(xiàn)有文獻(xiàn)報(bào)道最主要的SM-DNA加合物有以下四種：N7-（2-羥乙基硫代乙基）鳥嘌呤（N7-HETEG），雙[2-(N7-鳥嘌呤)]乙硫醚（Bis-G），O6-（2-羥乙基硫代乙基）鳥嘌呤（O6-HETEG），N3（-2-羥乙基硫代乙基）腺嘌呤（N3-HETEA）。這四種SM-DNA加合物的特點(diǎn)各不相同，如N7-HETEG的豐度最高，N3-HETEA是腺嘌呤上唯一烷基化產(chǎn)物，而根據(jù)堿基配對(duì)原則，Bis-G與O6-HETEG的反應(yīng)位點(diǎn)可直接破壞DNA雙鏈結(jié)構(gòu)等。因此同時(shí)分析這四類加合物就可以全面監(jiān)測(cè)DNA分子的烷基化程度，獲得芥子氣暴露后DNA的損傷及修復(fù)情況，進(jìn)而為評(píng)價(jià)救治方案提供可靠技術(shù)方法。 論文首先建立了同時(shí)檢測(cè)生物體內(nèi)不同功能組織以及尿液中四種SM-DNA加合物的同位素稀釋超高效液相色譜串級(jí)質(zhì)譜（ID-UPLC-MS/MS）分析方法；其次，建立合理的芥子氣皮膚染毒的實(shí)驗(yàn)動(dòng)物模型，取其組織器官和尿液；最后，利用建立的分析方法監(jiān)測(cè)SM-DNA加合物在生物體內(nèi)不同臟器的時(shí)效、量效分布，以便全面了解芥子氣在生物體內(nèi)從局部到整體的分布與代謝規(guī)律。論文共分六章。 第一章為前言，簡(jiǎn)要介紹了芥子氣的相關(guān)理化性質(zhì)，主要闡述其在體內(nèi)的代謝途徑和毒理機(jī)制的研究現(xiàn)狀，重點(diǎn)介紹芥子氣對(duì)DNA分子的損傷機(jī)理及其后續(xù)影響以及SM-DNA加合物的各種檢測(cè)手段，提出研究目標(biāo)和研究?jī)?nèi)容。 第二章重點(diǎn)建立了不同臟器中四種SM-DNA加合物的ID-UPLC-MS/MS同時(shí)分析測(cè)定方法。由于組織中SM-DNA連接在DNA分子上，為結(jié)合態(tài)加合物，導(dǎo)致樣品前處理過(guò)程相對(duì)復(fù)雜。 經(jīng)過(guò)一系列的優(yōu)化過(guò)程，該分析方法采用“酶解蛋白—酚氯仿抽提—乙醇沉淀”法提取DNA分子，回收率較高；甲酸加熱法水解堿基，利用ID-UPLC-MS/MS進(jìn)行分析。方法回收率為83-118%，檢測(cè)限為0.02-0.1ng/mL，定量限為0.05-0.2ng/mL。本章對(duì)組織勻漿、DNA提取到堿基水解等全過(guò)程進(jìn)行詳細(xì)、系統(tǒng)的優(yōu)化，建立了高效、穩(wěn)定的檢測(cè)方法，為下一章各臟器中DNA損傷的研究提供技術(shù)支持。 第三章建立了不同劑量下SD大鼠芥子氣皮膚染毒的動(dòng)物模型，對(duì)心臟、肝臟、脾臟、肺臟、胰臟、腎臟、全腦等主要組織中SM-DNA加合物進(jìn)行檢測(cè)，并對(duì)其中變化最顯著的組織進(jìn)行了系統(tǒng)的時(shí)效、量效關(guān)系研究，全面的考察了不同暴露劑量的條件下，芥子氣對(duì)不同功能的臟器組織中DNA分子的損傷情況及其自身修復(fù)的過(guò)程。 實(shí)驗(yàn)結(jié)果表明SM-DNA加合物與染毒劑量和時(shí)間之間存在良好的量效、時(shí)效相關(guān)性；第一次系統(tǒng)證實(shí)了體內(nèi)各臟器組織中Bis-G是豐度比例占第二位的DNA加合物（N7-HETEG，62.5-92.0%；Bis-G，7.9-37.0%；N3-HETEA，0.1-2.0%；O6-HETEG0.1%；），揭示芥子氣對(duì)DNA的損傷在前期研究中被嚴(yán)重低估；染毒劑量增加時(shí)，肺臟所受的損傷更為嚴(yán)重；肝臟可能并不是芥子氣損傷的主要器官；發(fā)現(xiàn)脂類含量高或被脂肪包裹的組織中SM-DNA加合物含量相對(duì)較高，提示我們芥子氣可能存在體內(nèi)“脂肪蓄積”的問題；芥子氣可以迅速通過(guò)血腦屏障，腦中脂類含量高，更易吸收并儲(chǔ)存親脂性的芥子氣原型分子，導(dǎo)致單位DNA形成加合物比例高。 第四章單獨(dú)考察了芥子氣對(duì)大鼠骨髓組織的損傷。骨髓組織是免疫細(xì)胞的產(chǎn)生地，實(shí)驗(yàn)將骨髓細(xì)胞按功能進(jìn)行分型，分別觀察了芥子氣中毒后不同功能骨髓細(xì)胞的應(yīng)答反應(yīng)，有助于從細(xì)胞層面上了解芥子氣對(duì)免疫系統(tǒng)的影響，并試圖以此結(jié)果部分解釋生物機(jī)體的中毒癥狀。 研究結(jié)果表明：通過(guò)對(duì)骨髓細(xì)胞中SM-DNA加合物進(jìn)行檢測(cè)，發(fā)現(xiàn)骨髓中Bis-G不僅豐度高，而且隨劑量增加其豐度比例甚至超過(guò)50%，成為最主要的DNA加合物，提示芥子氣對(duì)骨髓中DNA更嚴(yán)重的損傷；芥子氣染毒后，，大鼠骨髓中單核細(xì)胞呈現(xiàn)先劇烈增殖后快速抑制的趨勢(shì)，其計(jì)數(shù)變化幅度明顯，而粒細(xì)胞等多核細(xì)胞則緩慢增殖，同時(shí)免疫細(xì)胞的變化趨勢(shì)與動(dòng)物的中毒體征狀況具有高度的相關(guān)性；相同細(xì)胞計(jì)數(shù)下，單位單核或多核細(xì)胞中DNA加合物的含量近似，表明芥子氣對(duì)單核或多核細(xì)胞中DNA的損傷是等同或沒有偏好；而單位數(shù)量單核細(xì)胞中DNA加合物含量未呈現(xiàn)隨時(shí)間變化的趨勢(shì)，表明骨髓單核細(xì)胞中SM-DNA加合物含量的升高原因主要是由于細(xì)胞數(shù)量增加，再次證明芥子氣烷烴化作用的本質(zhì)。 第五章選取體內(nèi)代謝的最終載體—尿液作為檢測(cè)樣本，從整體輪廓層面分析SM-DNA的形成-修復(fù)-消失全過(guò)程，即觀察DNA損傷及修復(fù)的全身變化過(guò)程，與組織中結(jié)果相互補(bǔ)充、相互驗(yàn)證。 本部分開發(fā)了固相萃取新方法，用于尿液中四種SM-DNA加合物的同時(shí)提取。優(yōu)化ID-UPLC-MS/MS方法，回收率達(dá)到87-116%，檢測(cè)限為2-5pg/mL，定量限為5-10pg/mL，實(shí)現(xiàn)了對(duì)加合物的高靈敏檢測(cè)。建立了芥子氣皮膚染毒的動(dòng)物模型，并對(duì)尿液中SM-DNA加合物進(jìn)行分析檢測(cè)。結(jié)果顯示了良好的時(shí)效、量效關(guān)系曲線，并且獲得了尿液中四種SM-DNA加合物的真實(shí)比例關(guān)系。從宏觀上監(jiān)測(cè)DNA損傷的全身代謝輪廓，與組織中結(jié)果進(jìn)行比較，發(fā)現(xiàn)二者在時(shí)效關(guān)系以及加合物含量關(guān)系方面整體趨勢(shì)相同，可以實(shí)現(xiàn)良好的相互驗(yàn)證。由于尿液檢測(cè)無(wú)痛、無(wú)損，而且對(duì)于SM-DNA的檢測(cè)可以一定程度的反映機(jī)體DNA損傷情況，可作為芥子氣溯源分析以及損傷效應(yīng)監(jiān)測(cè)的理想檢測(cè)對(duì)象。 第六章是本論文建立方法的實(shí)際應(yīng)用。我們得到了四名意外接觸芥子氣患者的尿液，將已建立的固相萃取-ID-UPLC-MS/MS方法應(yīng)用于其尿液中四種SM-DNA加合物的同時(shí)定量檢測(cè)。檢測(cè)結(jié)果表明尿液中四種SM-DNA均可檢出，不僅更進(jìn)一步確證芥子氣染毒，而且發(fā)現(xiàn)加合物的含量與患者的暴露程度和臨床中毒癥狀相互吻合。上述結(jié)論證明SM-DNA加合物可以作為理想的芥子氣溯源性生物標(biāo)志物。
[Abstract]:Mustard gas, the full name of sulfur mustard in English (hereinafter referred to as SM, US military code HD), chemical formula C4H8Cl2S, is a double functional alkylating reagent and the most important one in the erosive chemical warfare agent. The preparation of mustard gas is simple, toxic and longer, is used in war several times, and still threatens world peace and people. Life safety is known as the "king of poison". However, the chemical nature of mustard gas determines its toxicity, and the research progress of its targeted detoxification drugs is slow in the world. Clinical treatment also treats it as a burn treatment. Therefore, the mechanism of the toxicological action of mustard gas is deeply studied and its special effect is screened and developed. Therapeutic drugs and clinical interventions have important practical significance, and these need to establish reliable methods for the detection and toxicological assessment of mustard gas.
For a long time, scholars at home and abroad have studied the mechanism of the toxicological action of mustard gas, and put forward a series of theories. Although the exact conclusion has not been reached yet, scholars in various countries generally agree that the alkylation damage of mustard gas to DNA molecules is the starting mechanism of its toxicological action and the material basis of cytotoxicity and biological toxicity. The endogenous and exogenous markers produced in the process of DNA alkylation damage and repair can be considered as an effective evaluation index for the damage and recovery of organisms after the exposure of mustard gas.
This paper uses a new method of analytical toxicology to detect the change trend of aging and quantitative effect of mustard gas and DNA alkylated adducts (hereinafter referred to as SM-DNA adducts) in vivo, to realize the real-time monitoring of the damage and repair process of DNA, so as to quantify the relationship between the material base of the toxicity of mustard gas cell and its toxicity. There are four main SM-DNA adducts in the current literature: N7- (2- hydroxyethyl thioethyl) guanine (N7-HETEG), double [2- (N7- guanine), Bis-G, O6- (2- hydroxyethyl thioethyl) guanine (O6-HETEG), N3 (-2- hydroxyethyl thioethyl) adenine. The characteristics of these four kinds of adducts are different. The abundance of 7-HETEG is the highest, N3-HETEA is the only alkylation product on adenine, and the reaction site of Bis-G and O6-HETEG can directly destroy the double chain structure of DNA according to the base pairing principle. Therefore, the analysis of these four kinds of adducts can monitor the degree of alkylation of DNA molecules and obtain the damage and repair of DNA after the exposure to mustard gas. It provides reliable technical methods for evaluating treatment plan.
The paper first established a method for the simultaneous detection of four SM-DNA adducts in different functional tissues and urine by isotopic dilution super high performance liquid chromatography tandem mass spectrometry (ID-UPLC-MS/MS). Secondly, a reasonable experimental animal model of the skin of mustard gas was established, and its tissues and urine were taken. Finally, the establishment of the model was established. The analysis method is used to monitor the distribution and distribution of SM-DNA adducts in different organs in the organism in order to understand the distribution and metabolism of mustard gas from local to whole in a comprehensive way. The paper is divided into six chapters.
In the first chapter, the related physical and chemical properties of mustard gas are briefly introduced. The research status of metabolic pathways and toxicological mechanisms in the body is mainly described. The damage mechanism and subsequent effects of mustard gas on DNA molecules as well as the various detection methods of SM-DNA adducts are introduced, and the research targets and research contents are presented.
The second chapter focuses on the establishment of ID-UPLC-MS/MS simultaneous determination method for four kinds of SM-DNA adducts in different organs. Because the SM-DNA connection in the tissue is on the DNA molecule, it is a combination of state adducts, which leads to the relative complexity of the sample pretreatment process.
After a series of optimization processes, the method used "enzyme hydrolysin - phenol chloroform extraction - ethanol precipitation" to extract DNA molecules, and the recovery rate was high. Acid heating method was used to hydrolyze base and ID-UPLC-MS/MS was used to analyze the method. The recovery rate was 83-118%, the detection limit was 0.02-0.1ng/mL, and the quantitative limit was 0.05-0.2ng/mL. to tissue homogenate. The whole process of DNA extraction to base hydrolysis is detailed and the system is optimized. A high efficient and stable detection method is established, which provides technical support for the research of DNA damage in the next chapter.
The third chapter established the animal model of SD rats with different doses of mustard gas, and tested the SM-DNA adducts in the main tissues such as heart, liver, spleen, lung, pancreas, kidney and whole brain, and studied the most significant changes in the relationship, and investigated the different exposure doses in a comprehensive way. The damage of mustard gas to DNA molecules in different functional organs and the process of self repair were studied.
The experimental results showed that there was a good dose effect and time correlation between the SM-DNA adducts and the dose and time. The first system confirmed that the Bis-G in the organs of the body was second DNA adducts (N7-HETEG, 62.5-92.0%; Bis-G, 7.9-37.0%; N3-HETEA, 0.1-2.0%; O6-HETEG0.1%;), and revealed that the mustard gas was to DNA. The damage was seriously underestimated in the previous study; the lung injury was more severe when the dose increased; the liver may not be the main organ of the mustard gas damage; it was found that the content of SM-DNA adducts in the fat wrapped tissues was relatively high, suggesting that our mustard gas may have "fat accumulation" in the body. The problem is that mustard gas can quickly pass through the blood brain barrier, the lipid content in the brain is high, and it is easier to absorb and store the lipophilic mustard gas prototype molecules, which leads to the high proportion of the unit DNA adducts.
The fourth chapter investigated the damage of the mustard gas to the bone marrow tissue of rats. The bone marrow tissue was the producing area of the immune cells. The experiment was used to classify the bone marrow cells according to the function. The response of different functional bone marrow cells after the mustard gas poisoning was observed respectively. This result partly explains the toxic symptoms of the organism.
The results showed that by detecting SM-DNA adducts in bone marrow cells, it was found that the abundance of Bis-G in bone marrow was not only high, but also increased with the abundance of more than 50%, which became the most important DNA adduct, suggesting that mustard gas was more serious to DNA in bone marrow. The trend of rapid inhibition after acute proliferation was obvious, while the multinucleated cells like granulocytes proliferated slowly, and the change trend of immune cells was highly correlated with the toxic signs of animals. Under the same cell count, the content of DNA adducts in unit mononuclear or multinuclear cells was similar, indicating that mustard gas is single. The damage of DNA in nuclear or multinuclear cells is the same or no preference, but the content of DNA adducts in unit number mononuclear cells does not show a tendency to change with time, indicating that the increase in the content of SM-DNA adducts in bone marrow mononuclear cells is mainly due to the increase in the number of cells, which proves the essence of the alkylation of mustard ananes again.
In the fifth chapter, the final carrier of metabolism in the body, urine as a sample, is used to analyze the whole process of the formation, repair and disappearance of SM-DNA from the overall outline level, that is to observe the whole body change process of DNA damage and repair, and to complement each other and verify the results of the tissue.
A new solid phase extraction method was developed for the simultaneous extraction of four SM-DNA adducts in urine. The ID-UPLC-MS/MS method was optimized, the recovery rate was up to 87-116%, the detection limit was 2-5pg/mL, and the quantitative limit was 5-10pg/mL. The high sensitivity test of the adduct was realized. The animal model of mustard skin skin skin dye was established and SM-DNA addition in urine was added to the urine. The results showed a good time limitation, a quantitative relationship curve, and a true proportion of four SM-DNA adducts in the urine. The overall metabolic profile of DNA damage was monitored from the macro level, and the results were compared with the results of the tissue. It was found that the overall trend of the aging relationship and the relationship of the adducts content was the same as the overall trend. Good mutual validation can be achieved. Since urine tests are painless and nondestructive, and the detection of SM-DNA can reflect the DNA damage to a certain extent, which can be used as an ideal target for the analysis of mustard gas traceability and the monitoring of damage effect.
The sixth chapter is the practical application of this method. We have obtained the urine of four accidental exposure to mustard gas. The established solid phase extraction -ID-UPLC-MS/MS method is applied to the simultaneous quantitative detection of four kinds of SM-DNA adducts in their urine. The results show that four kinds of SM-DNA in the urine can be detected, not only further corroborate the mustard It was found that the content of the adduct was consistent with the exposure degree of the patients and the symptoms of clinical poisoning. These conclusions have proved that the SM-DNA adduct can be used as an ideal biomarker for the origin of mustard gas.
【學(xué)位授予單位】：中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R994.3;R917

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