【摘要】：新煙堿類殺蟲劑是過去三十年中最重要的合成殺蟲劑。盡管和尼古丁在結(jié)構(gòu)和行為上相似,新煙堿類殺蟲劑起源于非逐個(gè)篩選,而是從結(jié)構(gòu)上進(jìn)行突破與嘗試進(jìn)而成為領(lǐng)導(dǎo)性的殺蟲劑。隨著新煙堿類殺蟲劑的廣泛應(yīng)用,它的毒性及其在環(huán)境中的影響變得日益重要。吡蟲啉是一種基于尼古丁的殺蟲劑,其作用類似于神經(jīng)毒素,屬于新煙堿類殺蟲劑。細(xì)胞培養(yǎng)模型被應(yīng)用于研究吡蟲啉對(duì)于哺乳細(xì)胞的毒性研究。應(yīng)用F11細(xì)胞系研究吡蟲啉對(duì)于神經(jīng)元細(xì)胞的毒性是一個(gè)可靠的方法。F11細(xì)胞系是由小鼠胚胎脊神經(jīng)末端細(xì)胞和大鼠成神經(jīng)瘤細(xì)胞系(N18TG2)雜合而成。該細(xì)胞系保留大鼠及小鼠染色質(zhì)及能夠合成及表達(dá)他們的同工酶,并能顯示出獨(dú)特的行為特性及表面受體的結(jié)合。 吡蟲啉在本研究中基本上很穩(wěn)定,在研究中沒有發(fā)現(xiàn)其代謝物。吡蟲啉對(duì)于處于血清培養(yǎng)48小時(shí)條件下的F11細(xì)胞系計(jì)數(shù)實(shí)驗(yàn)的半抑制率是1.21mM,因而其毒性對(duì)于該細(xì)胞而言較小。從MTT實(shí)驗(yàn)得到的吡蟲啉毒性說明F11細(xì)胞系的生長(zhǎng)及生存對(duì)于吡蟲啉更加敏感。另外,尼古丁對(duì)于實(shí)驗(yàn)條件下的F11細(xì)胞系的毒性較吡蟲啉更小 本文也研究了吡蟲啉對(duì)于F11細(xì)胞系形態(tài)學(xué)方面的毒性影響。經(jīng)過吡蟲啉培育的F11細(xì)胞系從形態(tài)學(xué)的角度上與對(duì)照實(shí)驗(yàn)有著較大的差異,顯示了吡蟲啉對(duì)于該神經(jīng)細(xì)胞的毒性影響。應(yīng)用微分相差干涉顯微鏡及抗β-tubulin標(biāo)記的熒光顯微鏡所觀測(cè)到的吡蟲啉培育后的F11細(xì)胞都出現(xiàn)了明顯的萎縮。在饑餓培育條件下應(yīng)用吡蟲啉處理的F11細(xì)胞亦能觀測(cè)到β-tubulin的聚集體。應(yīng)用小麥凝集素標(biāo)記的F11細(xì)胞膜的完整性在吡蟲啉的培育下被破壞,細(xì)胞膜的破壞很可能是高濃度的吡蟲啉所誘導(dǎo)的細(xì)胞凋亡表現(xiàn)之一。在吡蟲啉長(zhǎng)期處理下的F11細(xì)胞出現(xiàn)了細(xì)胞質(zhì)的濃縮,這也是細(xì)胞凋亡的表現(xiàn)之一 吡蟲啉干擾了F-肌動(dòng)蛋白的合成,并且由于細(xì)胞骨架的復(fù)雜性,這種對(duì)于F-肌動(dòng)蛋白形成的毒性影響很可能傳遞到其他的諸如肌動(dòng)蛋白結(jié)合蛋白、肌動(dòng)蛋白服務(wù)蛋白和肌動(dòng)蛋白捕捉蛋白上。對(duì)β2型nAChRs亞單位的表達(dá)研究表明,吡蟲啉能夠上調(diào)：lAChRs的合成。應(yīng)用吡蟲啉處理的F11細(xì)胞系能夠啟動(dòng)MAPK細(xì)胞信號(hào)傳導(dǎo)途徑及Nrf2細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)途徑。Nrf2能夠調(diào)控編碼抗氧化相關(guān)的酶及異質(zhì)物的解毒作用相關(guān)的酶。應(yīng)用磷酸化-p38蛋白的封閉劑能夠部分的降低吡蟲啉對(duì)于F11細(xì)胞的毒性影響。這說明了磷酸化-p38蛋白在吡蟲啉誘導(dǎo)下的F11細(xì)胞中確實(shí)參與到了細(xì)胞凋亡信號(hào)轉(zhuǎn)導(dǎo)中。尼古丁對(duì)于F11細(xì)胞系較低的毒性可能是因?yàn)槟峁哦?相對(duì)于毗蟲啉而言,在細(xì)胞體內(nèi)能夠誘導(dǎo)更多的Nrf2蛋白活性。 簡(jiǎn)言之,吡蟲啉在F11細(xì)胞中的行為可能如下所述。吡蟲啉結(jié)合在位于細(xì)胞膜的nAChRs受體后能過度激活該受體,因而通過受體形態(tài)上的變化能夠打開位于該受體上的離子通道。大量鈉離子和鉀離子的涌動(dòng)干擾了膜表面潛在的電位平衡。這將導(dǎo)致一些慢性毒性影響和胞內(nèi)活性氧濃度的增加,因而兩種不同的信號(hào)轉(zhuǎn)導(dǎo)通路被激活：Nrf2抗氧化細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)通路和p38細(xì)胞凋亡信號(hào)轉(zhuǎn)導(dǎo)通路。Nrf2解吸附位于細(xì)胞膜上的KEAP蛋白并轉(zhuǎn)移到細(xì)胞核內(nèi),結(jié)合于DNA鏈上的抗氧化響應(yīng)元件上,并激活下游編碼抗氧化酶及解毒酶的表達(dá),進(jìn)而提高細(xì)胞抗氧化能力。大量的活性氧富集于細(xì)胞內(nèi)同樣可能激活p38有絲分裂源蛋白激酶的活性。P38蛋白調(diào)控的細(xì)胞凋亡蛋白活性可能調(diào)控著吡蟲啉誘導(dǎo)的細(xì)胞凋亡途徑。 在本文中也仍然存在著一些問題需要更深一步的研究。首先,吡蟲啉對(duì)于哺乳動(dòng)物組織上的毒性影響亦然未知。吡蟲啉在細(xì)胞體內(nèi)及組織內(nèi)所表現(xiàn)的行為特性必然是不同的,吡蟲啉對(duì)于哺乳動(dòng)物組織方面的毒性影響對(duì)于其在環(huán)境中的地位及作用必然有著不可替代的作用。其次,限于實(shí)驗(yàn)室條件,一些有趣重要的研究無(wú)法開展。比如,全細(xì)胞電流檢測(cè)(?)AChRs的功能及高分辨率掃描隧道顯微鏡研究細(xì)胞亞結(jié)構(gòu),這些對(duì)于本課題而言都是有趣而重要的。最后,本文得出的結(jié)論需要更深一步的研究。P38蛋白封閉劑及N-乙酰半胱氨酸都能部分降低吡蟲啉對(duì)于F11細(xì)胞的毒性,說明細(xì)胞凋亡及抗氧化機(jī)制都在吡蟲啉處理下的細(xì)胞死亡中扮演重要的角色。然而,是否有其他的因素導(dǎo)致了細(xì)胞的死亡,及p38蛋白除開調(diào)控細(xì)胞凋亡途徑外,是否在F11細(xì)胞系中還扮演了其他的角色,這些亦然未知。 應(yīng)用F11細(xì)胞系研究吡蟲啉對(duì)于哺乳動(dòng)物神經(jīng)元的毒害作用,這種方法提供了一個(gè)不同但重要的途徑,盡管其中任然存在一些問題。鑒于目前所得到的一些有意義的研究結(jié)果,我們希望能在此領(lǐng)域進(jìn)行一些更深入的研究。
[Abstract]:New nicotinic insecticides are the most important synthetic insecticides in the past thirty years. Although they are similar to nicotine in structure and behavior, new nicotinic insecticides originate from one by one, but are structural breakthroughs and attempts to become leading insecticides. With the widespread use of new nicotinic insecticides, its toxicity and its effects are The influence of the environment is becoming increasingly important. Imidacloprid is an insecticide based on nicotine, which is similar to neurotoxin and is a new nicotinic insecticide. Cell culture model is used to study the toxicity of imidacloprid to mammalian cells. The use of F11 cell lines to study the toxicity of imidacloprid to neuron cells is a reliable method. The.F11 cell line is heterozygous from the terminal cells of the mouse embryonic spinal nerve and the rat neuroma cell line (N18TG2). This cell line keeps the chromatin of rats and mice and can synthesize and express their isozymes, and can show unique behavioral characteristics and binding of surface receptors.
Imidacloprid was basically stable in this study, and its metabolites were not found in the study. The semi inhibitory rate of imidacloprid for the F11 cell line count test under 48 hours of serum culture was 1.21mM, so its toxicity was smaller for this cell. The toxicity of the imidacloprid from the MTT test indicated the growth and survival of the F11 cell line. It is more sensitive to imidacloprid, and nicotine is less toxic to imidacloprid than the imidacloprid under the experimental conditions. F11 cell line is more sensitive to imidacloprid than that of imidacloprid.
The toxic effects of Imidacloprid on the morphology of F11 cell lines were also studied. The F11 cell line bred by imidacloprid had a large difference from the control experiment from the morphological point of view, showing the toxic effects of imidacloprid to the nerve cells. Differential phase phase interference microscope and anti beta -tubulin labeling fluorescence microscopy were used. The F11 cells of the imidacloprid (imidacloprid) cells observed by the mirror showed obvious atrophy. The F11 cells treated with imidacloprid under the condition of starvation were also able to observe the aggregates of beta -tubulin. The integrity of the F11 cell membrane labeled by wheat lectin was broken under the cultivation of imidacloprid, and the destruction of the cell membrane was likely to be high concentration. One of the apoptotic cells induced by imidacloprid (imidacloprid), one of the expression of cytoplasm in F11 cells under the long term treatment of imidacloprid, is also one of the manifestations of cell apoptosis.
Imidacloprid interferes with the synthesis of F- actin, and the toxic effects on F- actin formation due to the complexity of the cytoskeleton are likely to be transmitted to other actin binding proteins, actin service proteins and actin capture proteins. The expression of nAChRs subunits of beta 2 indicates that Imidacloprid Up to up: synthesis of lAChRs. Application of imidacloprid treated F11 cell lines can initiate MAPK cell signaling pathway and Nrf2 cell signal transduction pathway.Nrf2 can regulate the antidote related enzymes that encode antioxidant related enzymes and heterostructures. The application of phosphorylated -p38 protein blocking agents can partially reduce imidacloprid to F11 fines The toxicity of -p38 protein in the F11 cells induced by imidacloprid was indeed involved in the signal transduction of cell apoptosis. Nicotine may have a lower toxicity to the F11 cell line because of nicotine, which can induce more Nrf2 protein activity in the cell than the adhulline.
In short, the behavior of Imidacloprid in F11 cells may be described as follows. Imidacloprid binding to the nAChRs receptor in the cell membrane can excessively activate the receptor and thus open the ion channel located on the receptor by changes in the receptor morphology. The surge of sodium and potassium ions interferes with potential potential balance on the membrane surface. It will lead to some chronic toxic effects and increased intracellular ROS concentration, so two different signal transduction pathways are activated: Nrf2 antioxidant cell signal transduction pathway and p38 cell apoptosis signal transduction pathway.Nrf2 desorption of KEAP protein located on the cell membrane and transferred to the nucleus of the cell, combined with the antioxidant response on the DNA chain. On the components, the expression of anti oxidant oxidase and detoxification enzyme are activated and the antioxidant capacity of cells is enhanced. A large number of active oxygen enriched in cells may also activate the active.P38 protein kinase of p38 mitotic protein kinase, which may regulate the apoptotic pathway induced by imidacloprid.
In this paper, there are still some problems that need further research. First, the toxic effects of Imidacloprid on mammalian tissues are unknown. The behavior of Imidacloprid in the cells and tissues is necessarily different, and the toxic effects of Imidacloprid in the mammalian group are in the environment. Status and function must have an irreplaceable role. Secondly, some interesting and important research can not be carried out limited to laboratory conditions. For example, the function of the whole cell current detection (?) AChRs and the substructure of the high resolution scanning tunneling microscope (high resolution scanning tunneling microscope) are all interesting and important for this subject. Finally, the conclusion of this paper is the conclusion of this paper. A further study of.P38 protein sealers and N- acetylcysteine can partly reduce the toxicity of imidacloprid to F11 cells, indicating that cell apoptosis and antioxidant mechanisms play an important role in cell death under imidacloprid treatment. However, there are other factors that lead to cell death and the removal of p38 protein. It is also unknown whether the regulation of apoptosis pathway plays other roles in the F11 cell line.
Using the F11 cell line to study the toxicity of imidacloprid to mammalian neurons, this method provides a different but important approach, although there are some problems. In view of some of the useful results obtained, we hope to do some more in-depth research in this field.
【學(xué)位授予單位】：中國(guó)地質(zhì)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R114

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