本文選題：有機磷毒物　切入點：效應(yīng)標(biāo)志物　出處：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：有機磷毒物屬于一類含磷有機物,有機磷毒物依效能可分為神經(jīng)性毒劑(Nerve agents,NAs)和有機磷農(nóng)藥(Organophosphorus pesticides)。急性有機磷中毒的救治策略主要是利用阿托品進行對癥治療以及利用肟類化合物進行對因治療。而阿托品僅作用于毒蕈堿型受體,救治效果不佳,增大用藥劑量又會引起阿托品中毒;因此肟類化合物作為一種特異性解毒藥被越來越廣泛地應(yīng)用于急性有機磷中毒的救治。但肟類重活化劑無法進入中樞神經(jīng)系統(tǒng),且目前對其重活化效果存在一定爭議:一種觀點認(rèn)為肟類化合物通過重活化神經(jīng)或肌肉組織內(nèi)中毒乙酰膽堿酯酶(Acetylcholinesterase,AChE),從而恢復(fù)酶活性以及受損的身體機能;另一種觀點認(rèn)為肟類化合物在重活化過程中可與游離毒劑或與離去的磷酸(或膦酸)基團發(fā)生反應(yīng),形成一種膦�；恐虚g體,該中間體帶有季銨正電荷,更易攻擊帶有負(fù)電荷的酶活中心,故具有更強抑酶毒性,或許是造成肟類藥物重活化效果不佳甚至出現(xiàn)加深中毒現(xiàn)象的原因。目前對有機磷毒物中毒救治的研究工作多基于經(jīng)典的Ellman法等酶活測定法開展,然而Ellman法作為酶活測定的金標(biāo)準(zhǔn),依然存在一定的局限性,例如,AChE的測定易受丁酰膽堿酯酶(Butyrylcholinesterase,BChE)干擾,準(zhǔn)確度低;靈敏度低,當(dāng)中毒濃度低于20%時,無法準(zhǔn)確測定;同時測定酶活時,體系中其他膽堿酯酶抑制劑或重活化劑等藥物的存在會造成一定干擾,導(dǎo)致假陽性或假陰性結(jié)果;更為重要的是該方法特異性較差,無法分辨中毒類型,同時無法準(zhǔn)確描述AChE所處的狀態(tài)(活性態(tài)、中毒態(tài)、老化態(tài))。本課題研究建立了一種液相色譜-串級質(zhì)譜聯(lián)用(Liquid chromatography tandem mass spectrometry,LC-MS/MS)定量測定方法用于研究神經(jīng)性毒劑中毒特點與救治,該方法通過對毒物特異性加合肽段進行定量檢測,提供效應(yīng)酶中毒程度的數(shù)據(jù),同時能夠定量獲取效應(yīng)酶中毒后所處的不同狀態(tài),尤其是對于低劑量有機磷毒物暴露時,經(jīng)典Ellman法無法準(zhǔn)確獲得酶活性抑制率,而該方法對于抑制率小于1%的情況亦可進行準(zhǔn)確分析。本課題研究中,針對有機磷毒物中毒救治難題以及現(xiàn)有方法存在的諸多弊端,展開深入研究。本研究論文共分為四章:第一章:前言,對有機磷毒物及其中毒機理、中毒救治研究方法、抗毒藥物概況進行了介紹,并提出本論文的研究意義與目的。第二章:有機磷農(nóng)藥與AChE連接的基團多為二甲氧基膦�；鶊F(Dimethylphosphoryl,DMP)以及二乙氧基膦�；鶊F(Diethylphosphoryl,DEP),本章針對這兩類結(jié)構(gòu)特點制備了二甲基膦�；浑暮投一Ⅴ；浑�,在此基礎(chǔ)上利用課題組前期研究建立針對有機磷農(nóng)藥中毒救治研究的LC-MS/MS定量測定方法,對常見有機磷農(nóng)藥敵敵畏、敵百蟲、久效磷、對氧磷的中毒特點及重活化特點進行考察,有機磷農(nóng)藥的中毒過程相對神經(jīng)性毒劑較為緩慢,四種有機磷農(nóng)藥的中毒速率由快至慢依次為對氧磷、敵百蟲、敵敵畏、久效磷。對于同一種有機磷農(nóng)藥,不同重活化劑表現(xiàn)出不同的重活化效果:對于敵敵畏,雙復(fù)磷(Obidoxime,L-1656)的重活化效果更佳;對于敵百蟲,酰胺磷定(Asoxime,HI-6)的重活化效果更佳;對于久效磷,氯解磷定(Pralidoxime chloride,2-PAM)表現(xiàn)出顯著的重活化效果優(yōu)勢;對于對氧磷,三種重活化劑皆呈現(xiàn)良好的重活化效果,各重活化劑的抗毒效果呈現(xiàn)顯著差異,缺乏有效且廣譜的抗毒藥物;且通過比較發(fā)現(xiàn),二乙氧基膦�；卸久篙^二甲氧基膦�；卸久父妆恢鼗罨瘎┲鼗罨�。該評價工作對有機磷農(nóng)藥的合理使用、有機磷農(nóng)藥中毒的救治具有重要參考意義。第三章:針對有機磷農(nóng)藥救治效果不佳的原因,推測有機磷農(nóng)藥與重活化劑形成的膦�；�(Phosphoryl oxime,POX)是重活化效果的重要影響因素。為驗證推測,通過對氧磷與雙復(fù)磷在巴比妥緩沖液中直接反應(yīng)成功制備得到二者的反應(yīng)產(chǎn)物——DEP-obidoxime。在體外實驗中考察并比較對氧磷以及DEP-obidoxime對AChE的抑制作用差異,利用對氧磷以及DEP-obidoxime對AChE染毒,然后基于酶解-質(zhì)譜法的分析策略,將中毒酶經(jīng)胃蛋白酶酶解后產(chǎn)生含有加合位點的特異肽段,利用基于效應(yīng)標(biāo)志物的質(zhì)譜定量分析技術(shù)對特異性肽段進行定量檢測,實現(xiàn)對氧磷以及DEP-obidoxime中毒特點的考察,證實DEP-obidoxime具有較強的AChE抑制毒性,并進一步完成經(jīng)典重活化劑(氯解磷定、雙復(fù)磷、HI-6)抗毒效能評價工作,DEP-obidoxime膦�；腁ChE與對氧磷膦酰化的中毒酶可同樣被氯解磷定重活化,且重活化效果無顯著差異。同時半數(shù)有效濃度(Concentration for 50%of maximal effect,EC50)數(shù)據(jù)表明,3種經(jīng)典重活化劑對對氧磷或DEP-obidoxime膦酰化AChE的重活化效果由強至弱依次為:氯解磷定、HI-6、雙復(fù)磷。上述發(fā)現(xiàn)對于有機磷中毒的臨床救治用藥具有重要的參考價值。第四章:針對本課題組前期研究中發(fā)現(xiàn)肟類重活化劑會加深梭曼(Soman,GD)中毒的現(xiàn)象,推測肟類重活化劑作為抗毒藥物用于梭曼中毒救治過程中,可能與梭曼發(fā)生反應(yīng),形成對AChE抑制毒性更大的膦�；�,成為中毒加深的根源�；谶@一發(fā)現(xiàn),本章開展肟類重活化劑加深梭曼中毒機制的研究。首先,采用光譜法和氣相色譜法對神經(jīng)性毒劑梭曼與重活化劑的相互作用展開研究,研究發(fā)現(xiàn)肟類重活化劑雙復(fù)磷、氯解磷定以及HI-6可與梭曼明顯發(fā)生反應(yīng),而我所自主研發(fā)的叔胺類重活化劑L-1978、L-1655不與梭曼發(fā)生反應(yīng)。隨后在第三章研究工作的基礎(chǔ)上,成功制備得到梭曼與雙復(fù)磷以及氯解磷定的反應(yīng)產(chǎn)物PMPA-obidoxime、PMPA-pralidoxime。但由于梭曼中毒及老化十分迅速,無法有效觀察梭曼與膦酰化肟中毒及老化速率的差異,且因條件及技術(shù)限制,暫無法獲得梭曼光學(xué)異構(gòu)體單體,后續(xù)工作有待進一步開展。
[Abstract]:Organic phosphorus poison is a kind of organic phosphorous, organic phosphorus poison can be divided into nerve agents (Nerve agents, NAs in efficiency) and organophosphorus pesticides (Organophosphorus pesticides). The therapeutic strategy of acute organophosphate poisoning is the use of atropine for symptomatic treatment and due to treatment. The use of oximes and atropine only on muscarinic receptor, treatment effect, increase the dosage of atropine will cause poisoning; therefore oxime compounds as a specific antidote is more and more widely used in the treatment of acute organophosphorus poisoning. But oxime reactivators cannot enter the central nervous system, and the reactivative effect some controversy: a view of oximes by reactivative nerve or muscle tissue acetylcholinesterase (Acetylcholinesterase, AChE) poisoning, the recovery of enzyme activity As well as impaired physical function; another view is that in the process of oxime compounds with free agent or leave phosphoric acid in the heavy (or phosphonic acid) groups react to form a phosphonylation oxime intermediate, the intermediate with positively charged quaternary ammonium, easier to attack with negative charge activity center so, have a stronger inhibitory toxicity, may be caused by oxime drug reactivative effect or even cause poisoning. At present, deepen research work on organic phosphorus poisoning treatment based on Determination of the classical Ellman method to carry out live enzymes, enzyme activity determination method of Ellman as the gold standard, limitations still exist. Some, for example, AChE was susceptible to butyrylcholinesterase (Butyrylcholinesterase, BChE) interference, low sensitivity and low accuracy; when the concentration is below 20%, which can not be measured accurately; simultaneous determination of enzyme activity in the system. He is a cholinesterase inhibitor or reactivators and other drugs exist will cause some interference, leading to false positive or false negative results; more important is this method is not specific, and cannot distinguish the poisoning type, can not accurately describe the state that the AChE (active state, poisoning state, aging state). This research established a liquid chromatography / tandem mass spectrometry (Liquid chromatography tandem mass spectrometry, LC-MS/MS) is used to study the characteristics and treatment of nerve agent poisoning quantitative determination method, this method based on the specific and quantitative detection of toxic synthetic peptides, provide the effect of enzyme poisoning degree data, and to the varying state quantitatively get the effect of enzyme poisoning, especially for the low dose of organophosphorus poison exposure, the classical Ellman method cannot accurately obtain the enzyme activity inhibition rate, and the method for inhibiting rate of less than 1%. Condition can also be accurately analyzed. In this research, aiming at the disadvantages of organophosphorus poisoning and treatment problems of existing methods, in-depth research. This thesis is divided into four chapters: the first chapter: introduction of organic phosphorus poison and its mechanism of poisoning, poisoning and treatment methods, anti drug profiles are introduced. And put forward the significance and purpose of this paper. The second chapter: organophosphorus pesticide is connected with the AChE group for two methoxy phosphonic groups (Dimethylphosphoryl, DMP) and two ethoxylated phosphonic groups (Diethylphosphoryl, DEP), this chapter focuses on these two types of structure were prepared two methyl phosphonic acid eleven peptides and two ethyl phosphonylation eleven peptide, on the basis of previous studies to establish LC-MS/MS quantitative treatment of organophosphorus pesticide poisoning in the determination of common Organophosphorus Pesticide Dichlorvos, trichlorfon, Monocrotophos, inspect the poisoning characteristics and reactivative characteristics of phosphorus, organic phosphorus pesticide poisoning process relative to nerve agents is slow, poisoning rate of four kinds of organophosphorus pesticide from fast to slow in the paraoxon, trichlorfon, dichlorvos, monocrotophos. For the same kinds of organophosphorus pesticides different reactivators showed reactivative effect of different: for dichlorvos, obidoxime (Obidoxime, L-1656) better reactivative effect; for trichlorfon, HI-6 (Asoxime, HI-6) better reactivative effect; for monocrotophos, pralidoxime chloride (Pralidoxime chloride, 2-PAM) showed an advantage reactivative effect; for paraoxon, three kinds of reactivators are showing reactivative effect is good, showing significant difference in each reactivators antitoxic effect, lack of effective and broad-spectrum antiviral drugs; and it was found through comparison that two ethoxy phosphonylation poisoning with enzyme Two methoxy phosphonylation poisoning were more susceptible to the enzyme reactivators reactivative. Reasonable use of the evaluation work of organophosphorus pesticides, has important significance in the treatment of organophosphorus pesticide poisoning. The third chapter: the reasons for the organophosphorus pesticide treatment effect, that of organic phosphorus pesticide and phosphonylation oxime reactivative the formation of agent (Phosphoryl oxime POX) is an important factor influencing the reactivative effect. In order to verify that by paraoxon and obidoxime in barbital buffer was prepared by direct reaction of the success of the two reaction product DEP-obidoxime. in vitro experiments to investigate and compare the inhibitory effect of DEP-obidoxime on phosphorus and oxygen the differences in AChE, using paraoxon and DEP-obidoxime to AChE exposure, then enzyme analysis strategy based on mass spectrometry, poisoning the enzyme by pepsin produced containing specific peptide binding site, using the medium To mark the effect of mass spectrometry quantitative analysis technique for quantitative detection of specific peptide for the investigation of oxygen phosphorus and DEP-obidoxime poisoning, confirmed that DEP-obidoxime has strong inhibition of AChE toxicity, and further complete the classic reactivators (pralidoxime chloride, obidoxime, HI-6) antitoxic effectiveness evaluation, enzyme poisoning DEP-obidoxime phosphonylation AChE and paraoxon phosphonylation can also be pam-ci reactivative, and the activation effect was no significant difference. At the same time the median effective concentration (Concentration for 50%of maximal effect, EC50) data show that 3 kinds of classic reactivators reactivative effect of paraoxon or DEP-obidoxime phosphine acylated AChE were from strong to weak: pralidoxime chloride, HI-6, obidoxime. These findings have important reference value for the clinical treatment of drug organophosphate poisoning. The fourth chapter: according to the previous study Discovery of oxime reactivators will deepen Soman (Soman, GD) poisoning, presumably oxime reactivators as antidotes for Soman poisoning treatment process, may react with Soman, the formation of phosphonylation oxime on AChE inhibited the more toxic, poisoning become a source deepening. Based on this discovery in this chapter, the research development of oxime reactivators deepen Soman poisoning mechanism. Firstly, research the interaction by spectroscopy and gas chromatography of nerve agents Soman and reactivators, the study found that oxime reactivators cmo4, pralidoxime chloride, HI-6 and Soman obvious reaction I work and tertiary amine self-developed agent L-1978, L-1655 does not react with Soman. Then in the third chapter based on the research work, successfully prepared by Soman and obidoxime and pralidoxime chloride reaction products PMPA-obidoxime, PMPA-pralido Xime. but due to soman intoxication and aging rapidly, unable to effectively observe the differences of soman and phosphonylation oxime poisoning and aging rate, and because the conditions and restrictions, temporarily unable to obtain soman stereoisomers of monomer, the follow-up work need to be done.

【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R595.4

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