本文選題：離子液體 + HepG2細(xì)胞　； 參考：《江蘇大學(xué)》2017年碩士論文

【摘要】：因離子液體具有優(yōu)良的物化特性,目前已在各種工業(yè)生產(chǎn)如有機(jī)合成、生物催化及電化學(xué)等領(lǐng)域得到廣泛應(yīng)用,被認(rèn)為是“21世紀(jì)新型友好型溶劑”。離子液體廣泛使用的同時(shí),不可避免的排放或流失到環(huán)境中,對(duì)生物有機(jī)體甚至人類健康造成威脅,因此,離子液體的安全性及其對(duì)生態(tài)系統(tǒng)的毒性效應(yīng)成為人們研究的焦點(diǎn)。本文選用模式生物人類肝癌細(xì)胞(HepG2)和斑馬魚(yú)(Danio rerio)作為受試對(duì)象,系統(tǒng)研究了離子液體對(duì)人肝癌細(xì)胞Hep G2活性氧誘導(dǎo)作用、在斑馬魚(yú)體內(nèi)的分布積累和代謝以及對(duì)斑馬魚(yú)甲狀腺系統(tǒng)的干擾作用及機(jī)理。主要研究結(jié)果如下:1.不同濃度離子液體1-丁基-3-甲基-咪唑四氟硼酸鹽[BMIM]BF_4暴露HepG2細(xì)胞24 h后,MTT(噻唑藍(lán))實(shí)驗(yàn)結(jié)果發(fā)現(xiàn),在2.5,5,10 mM暴露組細(xì)胞活性顯著下降,明顯抑制細(xì)胞的生長(zhǎng)增殖,表現(xiàn)出顯著的細(xì)胞毒性;并誘導(dǎo)HepG2細(xì)胞產(chǎn)生過(guò)量的羥基自由基、超氧陰離子和總活性氧水平,從而導(dǎo)致細(xì)胞的氧化損傷。實(shí)時(shí)定量PCR結(jié)果表明nox2和nox4基因受到誘導(dǎo)表達(dá)而顯著上調(diào),提示這兩個(gè)基因可能是[BMIM]BF_4誘導(dǎo)HepG2細(xì)胞過(guò)量活性氧產(chǎn)生的關(guān)鍵基因;NADPH氧化酶途徑可能是[BMIM]BF_4誘導(dǎo)HepG2細(xì)胞產(chǎn)生過(guò)量活性氧的途徑之一。2.利用熒光氯化1,3-二(9-甲基蒽)咪唑鹽離子液體[DENMIM]C l的熒光特性,直觀、實(shí)時(shí)的觀察記錄了[DENMIM]Cl在斑馬魚(yú)體內(nèi)的累積、分布與代謝過(guò)程。結(jié)果顯示離子液體在斑馬魚(yú)幼魚(yú)體內(nèi)的積累呈現(xiàn)顯著的時(shí)間-劑量依賴性;通過(guò)血液循環(huán)及代謝過(guò)程,[DENMIM]Cl主要分布于肝臟、胃腸道和消化道中;在凈化階段,[DENMIM]Cl隨著時(shí)間延長(zhǎng)成功的被代謝及排泄出體外。3.將斑馬魚(yú)幼魚(yú)暴露于不同濃度的[BMIM]BF_4 10 d,20 d和30 d后,分析探討了[BMIM]BF_4對(duì)斑馬魚(yú)的下丘腦-垂體-甲狀腺軸(HPT軸)的干擾效應(yīng)及作用機(jī)理。結(jié)果顯示,[BMIM]BF_4暴露使幼魚(yú)體重、體長(zhǎng)顯著下降,生長(zhǎng)因子(CF)上升;不同濃度和時(shí)間的暴露造成幼魚(yú)體內(nèi)的甲狀腺激素水平失衡,導(dǎo)致T4水平下降,T3在暴露后20 d,30 d上升;在基因轉(zhuǎn)錄水平上檢測(cè)了[BMIM]BF_4對(duì)斑馬魚(yú)幼魚(yú)甲狀腺軸相關(guān)基因的干擾效應(yīng)。如下丘腦-垂體水平的crh、tsh,甲狀腺激素合成相關(guān)基因slc5a、tg、tpo上調(diào),這可能是由于T4水平的降低誘導(dǎo)HPT軸正反饋調(diào)節(jié),致使相關(guān)基因表達(dá)上調(diào)以維持穩(wěn)態(tài)造成的;而受體蛋白trα、trβ、脫碘酶dio2、轉(zhuǎn)運(yùn)蛋白ttr、代謝相關(guān)基因ugt1ab的異常表達(dá)可進(jìn)一步表明[BMIM]BF_4對(duì)斑馬魚(yú)幼魚(yú)甲狀腺軸的干擾作用,導(dǎo)致魚(yú)體內(nèi)甲狀腺激素水平紊亂從而影響其正常生理功能的發(fā)揮。
[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 偽 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.
【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：X171.5

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