本文選題：喹烯酮 + 凋亡��； 參考：《中國農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：喹烯酮是我國自主研發(fā)的一類新獸藥,其作為飼料添加劑廣泛應(yīng)用于畜牧水產(chǎn)養(yǎng)殖的生產(chǎn)實(shí)踐中,具有良好的抗菌促生長作用。上世紀(jì)九十年代一系列的常規(guī)毒理試驗(yàn)證明了喹烯酮作為飼料添加劑的安全性。但是進(jìn)入新世紀(jì)后,食品安全的重要性越來越受到關(guān)注。喹烯酮屬于喹VA啉類化合物,同一類中還包括喹乙醇、卡巴氧等。喹乙醇和卡巴氧都被證實(shí)具有顯著的毒性,與其擁有相同核心結(jié)構(gòu)的喹烯酮的安全性也受到了重新審視。最近本實(shí)驗(yàn)室對喹烯酮引起的細(xì)胞凋亡及其通路進(jìn)行了研究,對喹烯酮引起細(xì)胞的死亡機(jī)制有了初步了解。本研究擬從細(xì)胞程序性死亡的角度對喹烯酮作用下細(xì)胞死亡的分子機(jī)制做進(jìn)一步的探索。試驗(yàn)結(jié)果表明喹烯酮與ML-7聯(lián)用時,對HepG2細(xì)胞毒性增加,細(xì)胞凋亡率進(jìn)一步升高,線粒體膜電位消失的效應(yīng)增強(qiáng),外源性和內(nèi)源性凋亡通路都更加被激活。以上結(jié)果表明,喹烯酮通過凋亡引起細(xì)胞死亡的過程可以被ML-7加強(qiáng)。進(jìn)一步研究表明,ML-7刺激了喹烯酮引起的MAPKs信號通路,同時抑制了喹烯酮引起的Akt信號通路。并且,運(yùn)用MAPKs抑制劑和Akt激動劑之后的試驗(yàn)結(jié)果表明,Akt通絡(luò)和MAPKs通路都參與了喹烯酮引起的細(xì)胞凋亡。綜上所述,喹烯酮可以通過Akt通路和MAPKs通路調(diào)控凋亡的發(fā)生,具體機(jī)制為喹烯酮通過p38來誘發(fā)凋亡,而ERK, JNK和Akt通路則在此過程中抑制凋亡的發(fā)生。自噬又被稱為細(xì)胞Ⅱ型程序性死亡,GFP-LC3質(zhì)粒轉(zhuǎn)染喹烯酮處理的HepG2細(xì)胞發(fā)現(xiàn),喹烯酮使細(xì)胞中LC3聚集點(diǎn)增多,LC3的轉(zhuǎn)化率呈喹烯酮濃度依賴性和時間依賴性升高。結(jié)果表明喹烯酮引起了HepG2細(xì)胞發(fā)生自噬。接著,研究發(fā)現(xiàn)內(nèi)質(zhì)網(wǎng)應(yīng)激蛋白BiP和CHOP的表達(dá)量呈喹烯酮時間依賴性和濃度依賴性升高,并且sec24D、HerpUD、BiP的轉(zhuǎn)錄活性呈喹烯酮濃度依賴性升高。結(jié)果表明喹烯酮引起了HepG2細(xì)胞內(nèi)的內(nèi)質(zhì)網(wǎng)應(yīng)激。進(jìn)一步研究發(fā)現(xiàn),喹烯酮處理后,通過ATF6/DAPK1/MRLC的級聯(lián)反應(yīng)使mAtg9a的轉(zhuǎn)移加強(qiáng),從而促進(jìn)了自噬小體的生成。在干擾掉ATF6和DAPK1后,自噬標(biāo)志蛋白LC3的轉(zhuǎn)化率隨之降低。綜上所述,喹烯酮通過引起內(nèi)質(zhì)網(wǎng)應(yīng)激誘導(dǎo)了自噬通量的上升。線粒體自噬是一種具有底物特異性的選擇性自噬。本研究結(jié)果表明,線粒體自噬的核心調(diào)控蛋白parkin和PINK1在喹烯酮作用的HepG2細(xì)胞中呈濃度依賴性和時間依賴性降低；。線粒體膜蛋白COX IV蛋白表達(dá)降低、Tom20和Tim23的蛋白表達(dá)升高,與陽性對照CCCP產(chǎn)生的蛋白變化相同。結(jié)果表明,喹烯酮處理下的HepG2細(xì)胞發(fā)生了線粒體自噬。對線粒體自噬受體的western blot檢測顯示,在喹烯酮作用下OPTN變化不顯著,而TAX1BP1、FUNDC2表達(dá)量顯著升高。但是,在CCCP的作用下OPTN顯著升高,TAX1BP1顯著降低,FUNDC2變化不明顯。結(jié)果表明,喹烯酮作用下的HepG2細(xì)胞中線粒體自噬受體的變化趨勢與典型的線粒體自噬有很大不同。進(jìn)一步的試驗(yàn)運(yùn)用parkin-Flag高表達(dá)質(zhì)粒轉(zhuǎn)染HepG2細(xì)胞。結(jié)果顯示,喹烯酮和CCCP對parkin-Flag蛋白量都有顯著抑制。轉(zhuǎn)染質(zhì)粒后HepG2在喹烯酮和CCCP處理下的線粒體膜蛋白和自噬相關(guān)蛋白的變化趨勢都進(jìn)一步加強(qiáng)。并且,喹烯酮和CCCP都引起了HepG2細(xì)胞線粒體膜電位降低。但是,CCCP使HepG2細(xì)胞的線粒體數(shù)量顯著下降,而喹烯酮對線粒體數(shù)量的影響并不顯著,parkin轉(zhuǎn)染對結(jié)果影響也不明顯。結(jié)果表明,雖然喹烯酮能使線粒體膜電位下降,但是對線粒體數(shù)量沒有明顯影響。綜上所述,喹烯酮對線粒體的損傷作用雖然啟動了HepG2細(xì)胞中的線粒體自噬,但是并沒能造成線粒體數(shù)量的減少。從以上研究中可以得出結(jié)論,喹烯酮能引起HepG2細(xì)胞發(fā)生凋亡、自噬通量升高、以及線粒體自噬的起始。證明程序性細(xì)胞死亡在喹烯酮的毒性作用中發(fā)揮了重要作用。
[Abstract]:As a kind of new veterinary drug developed independently in China, it is widely used as feed additive in the production practice of animal husbandry and aquaculture. It has good antibacterial and growth promoting effect. In the 90s of last century, a series of conventional toxicological tests proved the safety of the addition agent of the feed. But after entering the new century, food safety The importance of the whole is becoming more and more concerned. Quinolone belongs to quinoline compounds. In the same class, quinolone, Kaba oxygen, and quinolone and Kaba oxygen have been proved to have significant toxicity. The safety of quinolone, which has the same core structure, has also been reconsidered. The cell withering caused by quinolone recently in our laboratory. This study intends to further explore the molecular mechanism of cell death under the action of programmed cell death. This study is to further explore the molecular mechanism of cell death under the action of the cell programmed cell death. The results show that when combined with ML-7, the toxicity of HepG2 cells increases and the rate of cell apoptosis is further increased. The effect of mitochondrial membrane potential was enhanced and the exogenous and endogenous apoptotic pathways were more activated. The above results showed that the process of cell death induced by apoptosis could be enhanced by ML-7. Further studies showed that ML-7 stimulated the MAPKs signaling pathway caused by the transports and inhibited the Akt signal caused by the ML-7. And, after the use of MAPKs inhibitors and Akt agonists, the results showed that both Akt collaterals and the MAPKs pathway were involved in the apoptosis induced by the Akt. To sum up, the mechanism of the apoptosis was mediated by the Akt pathway and the MAPKs pathway. The specific mechanism was that the apoptosis was induced by p38, while ERK, JNK and Akt pathways In this process, apoptosis is inhibited. Autophagy is also known as programmed cell type II death. GFP-LC3 plasmid transfected with HepG2 cells treated with OCT found that the LC3 aggregation point increased in the cells. The conversion rate of LC3 was dependent on the concentration and time dependence of the HepG2. The results showed that the HepG2 cells were autophagy. Then, it was found that the expression of endoplasmic reticulum stress protein BiP and CHOP showed a time dependence and concentration dependence of CHOP, and the transcriptional activity of sec24D, HerpUD, and BiP increased in the concentration dependence of the concentration of the endoplasmic reticulum. The results showed that the endoplasmic reticulum in the HepG2 cells was induced. The cascade of 6/DAPK1/MRLC enhanced the transfer of mAtg9a, thereby promoting the formation of autophagic bodies. The conversion rate of autophagy LC3 was reduced after interfering with ATF6 and DAPK1. In summary, the induction of autophagic flux was induced by endoplasmic reticulum stress. The results of this study showed that the core regulatory proteins parkin and PINK1 of mitochondrial autophagy were both concentration dependent and time-dependent in the HepG2 cells acting on the function of the PINK1; the expression of mitochondrial membrane protein COX IV protein was reduced, the protein expression of Tom20 and Tim23 increased, and the protein produced by the positive CCCP was the same. Western blot detection of autophagic receptors in HepG2 cells showed that OPTN changes were not significant under the action of quinolone, and the expression of TAX1BP1 and FUNDC2 increased significantly. However, the OPTN increased significantly under the action of CCCP, TAX1BP1 decreased significantly, and FUNDC2 changes were not obvious. The results showed that the FUNDC2 changes were not obvious. The results showed quinene The change trend of mitochondrial autophagy receptor in HepG2 cells was very different from that of typical mitochondrial autophagy. Further experiments used parkin-Flag high expression plasmid to transfect HepG2 cells. The results showed that the amount of parkin-Flag protein was significantly inhibited by the parkin-Flag and the transfected plasmid. The transfected plasmid was treated with the treatment of ketonone and CCCP. The change trend of mitochondrial membrane protein and autophagy related protein was further strengthened. Furthermore, the mitochondrial membrane potential decreased in HepG2 cells. However, CCCP decreased the number of mitochondria in HepG2 cells, and the effect of parkin on the number of mitochondria was not obvious, and the effect of parkin transfection on the results was not obvious. It was shown that although the membrane potential of the mitochondrial membrane decreased, but it had no obvious effect on the number of mitochondria. In summary, the damage of the mitochondria to mitochondria started the mitochondrial autophagy in HepG2 cells, but it did not cause a decrease in the number of mitochondria. Apoptosis, increased autophagy and initiation of mitochondrial autophagy have demonstrated that programmed cell death plays an important role in the toxicity of the compound.
【學(xué)位授予單位】：中國農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S859.8

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