吡虫啉对小鼠神经细胞的毒性研究

发布时间：2018-08-09 15:06

【摘要】：新烟碱类杀虫剂是过去三十年中最重要的合成杀虫剂。尽管和尼古丁在结构和行为上相似,新烟碱类杀虫剂起源于非逐个筛选,而是从结构上进行突破与尝试进而成为领导性的杀虫剂。随着新烟碱类杀虫剂的广泛应用,它的毒性及其在环境中的影响变得日益重要。吡虫啉是一种基于尼古丁的杀虫剂,其作用类似于神经毒素,属于新烟碱类杀虫剂。细胞培养模型被应用于研究吡虫啉对于哺乳细胞的毒性研究。应用F11细胞系研究吡虫啉对于神经元细胞的毒性是一个可靠的方法。F11细胞系是由小鼠胚胎脊神经末端细胞和大鼠成神经瘤细胞系(N18TG2)杂合而成。该细胞系保留大鼠及小鼠染色质及能够合成及表达他们的同工酶,并能显示出独特的行为特性及表面受体的结合。吡虫啉在本研究中基本上很稳定,在研究中没有发现其代谢物。吡虫啉对于处于血清培养48小时条件下的F11细胞系计数实验的半抑制率是1.21mM,因而其毒性对于该细胞而言较小。从MTT实验得到的吡虫啉毒性说明F11细胞系的生长及生存对于吡虫啉更加敏感。另外,尼古丁对于实验条件下的F11细胞系的毒性较吡虫啉更小本文也研究了吡虫啉对于F11细胞系形态学方面的毒性影响。经过吡虫啉培育的F11细胞系从形态学的角度上与对照实验有着较大的差异,显示了吡虫啉对于该神经细胞的毒性影响。应用微分相差干涉显微镜及抗β-tubulin标记的荧光显微镜所观测到的吡虫啉培育后的F11细胞都出现了明显的萎缩。在饥饿培育条件下应用吡虫啉处理的F11细胞亦能观测到β-tubulin的聚集体。应用小麦凝集素标记的F11细胞膜的完整性在吡虫啉的培育下被破坏,细胞膜的破坏很可能是高浓度的吡虫啉所诱导的细胞凋亡表现之一。在吡虫啉长期处理下的F11细胞出现了细胞质的浓缩,这也是细胞凋亡的表现之一吡虫啉干扰了F-肌动蛋白的合成,并且由于细胞骨架的复杂性,这种对于F-肌动蛋白形成的毒性影响很可能传递到其他的诸如肌动蛋白结合蛋白、肌动蛋白服务蛋白和肌动蛋白捕捉蛋白上。对β2型nAChRs亚单位的表达研究表明,吡虫啉能够上调：lAChRs的合成。应用吡虫啉处理的F11细胞系能够启动MAPK细胞信号传导途径及Nrf2细胞信号转导途径。Nrf2能够调控编码抗氧化相关的酶及异质物的解毒作用相关的酶。应用磷酸化-p38蛋白的封闭剂能够部分的降低吡虫啉对于F11细胞的毒性影响。这说明了磷酸化-p38蛋白在吡虫啉诱导下的F11细胞中确实参与到了细胞凋亡信号转导中。尼古丁对于F11细胞系较低的毒性可能是因为尼古丁,相对于毗虫啉而言,在细胞体内能够诱导更多的Nrf2蛋白活性。简言之,吡虫啉在F11细胞中的行为可能如下所述。吡虫啉结合在位于细胞膜的nAChRs受体后能过度激活该受体,因而通过受体形态上的变化能够打开位于该受体上的离子通道。大量钠离子和钾离子的涌动干扰了膜表面潜在的电位平衡。这将导致一些慢性毒性影响和胞内活性氧浓度的增加,因而两种不同的信号转导通路被激活：Nrf2抗氧化细胞信号转导通路和p38细胞凋亡信号转导通路。Nrf2解吸附位于细胞膜上的KEAP蛋白并转移到细胞核内,结合于DNA链上的抗氧化响应元件上,并激活下游编码抗氧化酶及解毒酶的表达,进而提高细胞抗氧化能力。大量的活性氧富集于细胞内同样可能激活p38有丝分裂源蛋白激酶的活性。P38蛋白调控的细胞凋亡蛋白活性可能调控着吡虫啉诱导的细胞凋亡途径。在本文中也仍然存在着一些问题需要更深一步的研究。首先,吡虫啉对于哺乳动物组织上的毒性影响亦然未知。吡虫啉在细胞体内及组织内所表现的行为特性必然是不同的,吡虫啉对于哺乳动物组织方面的毒性影响对于其在环境中的地位及作用必然有着不可替代的作用。其次,限于实验室条件,一些有趣重要的研究无法开展。比如,全细胞电流检测(?)AChRs的功能及高分辨率扫描隧道显微镜研究细胞亚结构,这些对于本课题而言都是有趣而重要的。最后,本文得出的结论需要更深一步的研究。P38蛋白封闭剂及N-乙酰半胱氨酸都能部分降低吡虫啉对于F11细胞的毒性,说明细胞凋亡及抗氧化机制都在吡虫啉处理下的细胞死亡中扮演重要的角色。然而,是否有其他的因素导致了细胞的死亡,及p38蛋白除开调控细胞凋亡途径外,是否在F11细胞系中还扮演了其他的角色,这些亦然未知。应用F11细胞系研究吡虫啉对于哺乳动物神经元的毒害作用,这种方法提供了一个不同但重要的途径,尽管其中任然存在一些问题。鉴于目前所得到的一些有意义的研究结果,我们希望能在此领域进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.
【学位授予单位】：中国地质大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R114

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