离子液体对人肝癌细胞HepG2活性氧水平和斑马鱼的毒性影响及机理研究

发布时间：2018-06-28 20:07

本文选题：离子液体 + HepG2细胞　；参考：《江苏大学》2017年硕士论文

【摘要】：因离子液体具有优良的物化特性,目前已在各种工业生产如有机合成、生物催化及电化学等领域得到广泛应用,被认为是“21世纪新型友好型溶剂”。离子液体广泛使用的同时,不可避免的排放或流失到环境中,对生物有机体甚至人类健康造成威胁,因此,离子液体的安全性及其对生态系统的毒性效应成为人们研究的焦点。本文选用模式生物人类肝癌细胞(HepG2)和斑马鱼(Danio rerio)作为受试对象,系统研究了离子液体对人肝癌细胞Hep G2活性氧诱导作用、在斑马鱼体内的分布积累和代谢以及对斑马鱼甲状腺系统的干扰作用及机理。主要研究结果如下:1.不同浓度离子液体1-丁基-3-甲基-咪唑四氟硼酸盐[BMIM]BF_4暴露HepG2细胞24 h后,MTT(噻唑蓝)实验结果发现,在2.5,5,10 mM暴露组细胞活性显著下降,明显抑制细胞的生长增殖,表现出显著的细胞毒性;并诱导HepG2细胞产生过量的羟基自由基、超氧阴离子和总活性氧水平,从而导致细胞的氧化损伤。实时定量PCR结果表明nox2和nox4基因受到诱导表达而显著上调,提示这两个基因可能是[BMIM]BF_4诱导HepG2细胞过量活性氧产生的关键基因;NADPH氧化酶途径可能是[BMIM]BF_4诱导HepG2细胞产生过量活性氧的途径之一。2.利用荧光氯化1,3-二(9-甲基蒽)咪唑盐离子液体[DENMIM]C l的荧光特性,直观、实时的观察记录了[DENMIM]Cl在斑马鱼体内的累积、分布与代谢过程。结果显示离子液体在斑马鱼幼鱼体内的积累呈现显著的时间-剂量依赖性;通过血液循环及代谢过程,[DENMIM]Cl主要分布于肝脏、胃肠道和消化道中;在净化阶段,[DENMIM]Cl随着时间延长成功的被代谢及排泄出体外。3.将斑马鱼幼鱼暴露于不同浓度的[BMIM]BF_4 10 d,20 d和30 d后,分析探讨了[BMIM]BF_4对斑马鱼的下丘脑-垂体-甲状腺轴(HPT轴)的干扰效应及作用机理。结果显示,[BMIM]BF_4暴露使幼鱼体重、体长显著下降,生长因子(CF)上升;不同浓度和时间的暴露造成幼鱼体内的甲状腺激素水平失衡,导致T4水平下降,T3在暴露后20 d,30 d上升;在基因转录水平上检测了[BMIM]BF_4对斑马鱼幼鱼甲状腺轴相关基因的干扰效应。如下丘脑-垂体水平的crh、tsh,甲状腺激素合成相关基因slc5a、tg、tpo上调,这可能是由于T4水平的降低诱导HPT轴正反馈调节,致使相关基因表达上调以维持稳态造成的;而受体蛋白trα、trβ、脱碘酶dio2、转运蛋白ttr、代谢相关基因ugt1ab的异常表达可进一步表明[BMIM]BF_4对斑马鱼幼鱼甲状腺轴的干扰作用,导致鱼体内甲状腺激素水平紊乱从而影响其正常生理功能的发挥。
[Abstract]:Due to their excellent physicochemical properties, ionic liquids have been widely used in various industrial production fields such as organic synthesis, biocatalysis and electrochemistry. They are considered as "new friendly solvents in the 21st century". With the widespread use of ionic liquids, it is inevitable to discharge or drain into the environment, which poses a threat to biological organisms and even human health. Therefore, the safety of ionic liquids and their toxic effects on ecosystems have become the focus of research. In this study, human hepatoma cell line (HepG2) and Danio rerio were used as experimental subjects to study the effects of ionic liquid on the induction of reactive oxygen species (Ros) in human hepatoma cell line HepG2. The distribution, accumulation and metabolism of zebrafish and its interference with the thyroid system of zebrafish and its mechanism. The main results are as follows: 1. Different concentrations of ionic liquid 1- Ding Ji -3-methyl-imidazolium tetrafluoroborate [BMIM] BF4 were exposed to HepG2 cells for 24 h. The results of MTT assay showed that the activity of HepG2 cells was significantly decreased and the proliferation of HepG2 cells was inhibited after exposure to 2.5 ~ 510mm. It showed significant cytotoxicity and induced excessive hydroxyl radical, superoxide anion and total reactive oxygen species in HepG2 cells, which resulted in oxidative damage of HepG2 cells. The results of real-time quantitative PCR showed that nox2 and nox4 genes were significantly up-regulated by induced expression. These two genes may be the key gene of [BMIM] BF4 to induce excessive reactive oxygen species production in HepG2 cells. The NADPH oxidase pathway may be one of the pathways of [BMIM] BF4 inducing excessive reactive oxygen species production in HepG2 cells. The accumulation, distribution and metabolism of [DENMIM] Cl in zebrafish were recorded by using the fluorescence characteristics of [DENMIM] C1, a fluorescent ionic liquid containing 1 / 3- bis (9-methylanthracene) imidazolium chloride. The results showed that the accumulation of ionic liquids in juvenile zebrafish was time-dose dependent, and [DENMIM] Cl was mainly distributed in liver, gastrointestinal tract and digestive tract through blood circulation and metabolism. In the purification stage, [DENMIM] Cl was successfully metabolized and excreted out of vitro. After the juvenile zebrafish were exposed to different concentrations of [BMIM] BF for 20 days and 30 days, the interference effect and mechanism of [BMIM] BF4 on the hypothalamus-pituitary-thyroid axis (HPT axis) of zebrafish were analyzed. The results showed that [BMIM] BF4 exposure significantly decreased the body weight, body length and growth factor (CF) of young fish, and that exposure of different concentrations and time resulted in the imbalance of thyroid hormone level in juvenile fish, which led to the decrease of T4 level and the increase of T3 level 20 days after exposure. The interference effect of [BMIM] BF4 on thyroid axis related genes in juvenile zebrafish was detected at the level of gene transcription. For example, the hypothalamus-pituitary crhtshand thyroid hormone synthesis related gene slc5atgtpo is up-regulated, which may be due to the decrease of T4 level inducing positive feedback regulation of HPT axis, resulting in the up-regulation of gene expression to maintain homeostasis. The abnormal expression of receptor protein tr 伪 tr 尾, deiodinase dio2, transporter Ttrand and metabolism-related gene ugt1ab may further indicate the interference of [BMIM] BF4 on the thyroid axis of juvenile zebrafish. The disorder of thyroid hormone level in fish affects its normal physiological function.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：X171.5

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