發(fā)布時間：2018-04-05 08:20

  本文選題：氟苯尼考　切入點：毒副作用　出處：《山東農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：隨著我國畜牧養(yǎng)殖規(guī)模的不斷擴大,畜禽疫病防控的壓力日益增大,多病原混合感染特別是細菌繼發(fā)感染現(xiàn)象較為普遍,大量抗生素被廣泛應(yīng)用于畜禽疾病的預(yù)防及獸醫(yī)臨床治療,并在飼料中被大量添加以提高飼料轉(zhuǎn)化率或達到促生長的目的。在此背景下,畜禽生產(chǎn)中濫用和亂用抗菌藥物的現(xiàn)象十分普遍,這給人畜健康和細菌病的防控帶來了巨大挑戰(zhàn)。氟苯尼考(FLO)是一種獸醫(yī)臨床專用的新型廣譜抗菌藥物,目前已成為畜牧及水產(chǎn)養(yǎng)殖過程中使用范圍最廣、使用量最大的抗菌藥物之一。“內(nèi)共生學(xué)說”指出真核生物的線粒體起源于古細菌,古細菌寄生于原始生物后隨著長時間的互利共生與演化過程而演變成了細胞線粒體,因此線粒體核糖體在結(jié)構(gòu)和功能上與細菌核糖體具有較高的相似性。基于此,真核細胞線粒體的核糖體便很容易受到特定抗菌藥物的干擾,并給動物機體或細胞帶來嚴重的毒副作用。FLO能夠與細菌核糖體A位點緊密結(jié)合而抑制肽酰轉(zhuǎn)移酶的活性,最終通過影響蛋白質(zhì)的合成而殺滅細菌,同時也會影響真核細胞線粒體蛋白質(zhì)的合成。隨著FLO的大量應(yīng)用,關(guān)于其毒副作用尤其是造血免疫毒性和胚胎毒性的報道也越來越多,但都基于FLO對機體生理機能或組織器官功能的影響觀察或檢測,尚未見到在細胞分子水平上研究FLO毒副作用的報道。為明確FLO對線粒體結(jié)構(gòu)與功能的具體影響,及其對細胞存活、細胞增殖及細胞穩(wěn)態(tài)的毒副作用,本研究以成纖維細胞為研究對象,在細胞及分子水平上研究了FLO對線粒體結(jié)構(gòu)與功能的影響,以及線粒體損傷對細胞增殖、細胞存活及線粒體自噬的影響及其機制,從分子水平揭示了FLO致成纖維細胞毒性的具體機制。一、FLO誘導(dǎo)線粒體結(jié)構(gòu)與功能損傷線粒體作為真核細胞內(nèi)進行有氧呼吸的主要場所,其結(jié)構(gòu)和功能的完整對細胞的生命活動來說至關(guān)重要。本研究首先聚焦在FLO對線粒體結(jié)構(gòu)與功能的影響方面,選用不同劑量的FLO(0.4、0.1、0.025 mg/m L)處理細胞48 h后,主要進行以下實驗:(1)Western blot分別檢測FLO對線粒體核糖體編碼的蛋白質(zhì)Cox I及細胞質(zhì)核糖體編碼的蛋白質(zhì)白Cox IV的表達水平的影響;(2)透射電鏡檢查FLO對細胞內(nèi)線粒體數(shù)量和形態(tài)結(jié)構(gòu)的影響;(3)流式細胞術(shù)分別檢測FLO對線粒體膜電位及細胞內(nèi)活性氧簇(ROS)生成量的影響;(4)生化方法分別測定FLO對線粒體呼吸鏈復(fù)合體I、II及IV的催化活性及細胞內(nèi)ATP生成量的影響。結(jié)果表明:FLO能夠顯著抑制由線粒體核糖體翻譯的線粒體蛋白如Cox I的表達,進而顯著下調(diào)線粒體呼吸鏈復(fù)合體I及IV的活性(p0.01)、顯著降低線粒體膜電位(p0.05或p0.01)及胞內(nèi)ATP的水平(p0.05或p0.01)并提高ROS的生成量;從結(jié)構(gòu)上來說,FLO處理后的細胞內(nèi)出現(xiàn)了腫脹、空泡化、嵴消失的損傷線粒體。以上結(jié)果表明線粒體正常的結(jié)構(gòu)和生理功能受到了FLO的嚴重影響。二、FLO誘導(dǎo)的線粒體功能障礙抑制細胞增殖活性正常線粒體在細胞周期調(diào)控和細胞增殖發(fā)育等方面具有重要作用,鑒于FLO可以造成明顯的線粒體功能障礙,我們隨后從以下幾方面研究了FLO對細胞存活、細胞周期和增殖調(diào)控的影響:(1)不同劑量FLO分別處理細胞12 h、24 h、36 h、48 h、60 h及72 h,通過細胞計數(shù)研究細胞增殖曲線;(2)CCK-8還原法測定不同劑量FLO處理細胞24 h、48 h及72 h對細胞增殖活性的影響;(3)Ed U摻入實驗檢測不同劑量FLO處理48 h對細胞DNA復(fù)制活性的影響;(4)流式細胞術(shù)檢測不同劑量FLO對細胞周期分布的影響;(5)流式細胞術(shù)及caspase-3活性分析實驗檢測FLO對細胞凋亡的影響;(6)乳酸脫氫酶(LDH)釋放實驗檢測FLO對細胞死亡率的影響;(7)Western blot檢測FLO對細胞內(nèi)增殖相關(guān)信號通路活化狀態(tài)的影響。結(jié)果表明:(1)FLO能導(dǎo)致與劑量有關(guān)的細胞活力降低,表現(xiàn)在細胞絕對數(shù)量的下降和增殖活力的降低(p0.01);(2)Ed U摻入試驗及細胞周期檢測結(jié)果表明FLO能顯著降低發(fā)生DNA復(fù)制的細胞比例,并使細胞周期發(fā)生G0/G1期阻滯(p0.05或p0.01);(3)選用D-半乳糖培養(yǎng)基迫使細胞只能通過線粒體呼吸鏈供能以便于使線粒體毒性物質(zhì)對線粒體的毒副作用表現(xiàn)得更為明顯,給予FLO后發(fā)現(xiàn)多種細胞表現(xiàn)出了更為嚴重的細胞活力抑制作用(p0.01),表明FLO對線粒體呼吸鏈的損傷與其所致的細胞活力下降直接相關(guān);(4)FLO對細胞凋亡率和壞死率無明顯影響(p0.05),說明FLO所致細胞活力下降僅與細胞增殖減緩有關(guān),與細胞死亡則無明顯關(guān)聯(lián);(5)從機制上來說,FLO活化了AMPK-m TOR-p70S6K通路,即FLO使活化的AMPK(磷酸化)表達上調(diào)、磷酸化的m TOR及磷酸化的p70S6K表達下調(diào),同時ROS-p53-p21通路也被活化,AMPK特異性抑制劑Compound C和抗氧化劑NAC分別能夠特異性抑制上述相應(yīng)通路的活化并提高被FLO抑制的細胞增殖活性(p0.05或p0.01)。三、FLO導(dǎo)致細胞內(nèi)損傷線粒體清除障礙及細胞衰老發(fā)生線粒體自噬對損傷線粒體的清除和細胞穩(wěn)態(tài)的維持至關(guān)重要,鑒于FLO可以造成明顯的線粒體功能障礙,我們隨后從以下幾方面研究了FLO對細胞內(nèi)損傷線粒體的清除過程即線粒體自噬的影響:(1)Western blot檢測FLO對胞內(nèi)線粒體質(zhì)量(線粒體內(nèi)膜蛋白Tim23水平)的影響;(2)使用線粒體特異性探針Mito Tracker Green染色線粒體,并用流式細胞術(shù)檢測FLO對細胞內(nèi)線粒體數(shù)量的影響;(3)使用自噬相關(guān)蛋白LC3的特異性抗體染色結(jié)合共聚焦顯微鏡檢查FLO處理的細胞內(nèi)自噬點的形成情況;(4)Western blot檢測FLO對自噬相關(guān)蛋白LC3B II/I及p62的表達水平,以評估細胞自噬水平;(5)衰老相關(guān)β-半乳糖苷酶染色檢測FLO處理8 d所造成的的損傷線粒體累積對細胞衰老水平的影響;(6)Western blot檢測FLO對招募至損傷線粒體的線粒體自噬起始關(guān)鍵蛋白Parkin及p62水平的影響,結(jié)合ROS清除劑NAC研究p53蛋白對Parkin向線粒體轉(zhuǎn)位的影響;(7)Mito Tracker Green染色結(jié)合流式細胞術(shù)檢測NAC對線粒體自噬或損傷線粒體清除功能的調(diào)節(jié)作用。結(jié)果表明:(1)細胞自噬功能在FLO處理的細胞中處于抑制狀態(tài),表現(xiàn)在自噬相關(guān)蛋白LC3B II下降和p62蛋白表達水平上升,細胞內(nèi)LC3熒光聚點的數(shù)量在不同處理組間無顯著差異;(2)胞內(nèi)線粒體內(nèi)膜蛋白Tim23的水平在FLO給藥后輕微升高,FLO處理組的Mitotracker Green熒光值顯著升高,表明FLO處理后損傷的線粒體并不能通過自噬有效清除,而是堆積在細胞內(nèi);(3)Parkin蛋白從胞漿被招募到線粒體是線粒體自噬的關(guān)鍵環(huán)節(jié),本研究發(fā)現(xiàn)FLO處理后線粒體膜上Parkin蛋白的量及其招募的p62蛋白的量都未升高,表明FLO所致線粒體自噬異常與Parkin蛋白的線粒體轉(zhuǎn)位障礙有關(guān);(4)使用NAC和FLO共處理細胞后發(fā)現(xiàn)Parkin蛋白線粒體轉(zhuǎn)運障礙能夠得到緩解,結(jié)合先前p53蛋白抑制Parkin蛋白線粒體轉(zhuǎn)位的報道可知,FLO處理活化了ROS-p53通路并通過抑制Parkin蛋白的線粒體轉(zhuǎn)位發(fā)揮線粒體自噬抑制效應(yīng),進而使損傷的線粒體得不到有效清除并最終導(dǎo)致細胞衰老的發(fā)生。本研究從分子水平揭示了氟苯尼考致成纖維細胞毒性的具體機制,研究結(jié)果對于明確線粒體毒性藥物的毒性機制具有重要借鑒意義,有助于指導(dǎo)氟苯尼考在畜牧及水產(chǎn)養(yǎng)殖中的合理應(yīng)用;同時對靶向線粒體的抗腫瘤藥物研發(fā)及新一代抗生素的線粒體安全性評價具有借鑒意義。
[Abstract]:Along with our country animal husbandry continues to expand the scale of animal disease prevention and control, increasing pressure, multiple pathogens infection especially secondary bacterial infection is a common phenomenon, a large number of antibiotics have been widely used in livestock and poultry disease prevention and veterinary clinical treatment, and in the feed was added in order to improve the feed conversion rate reached the purpose of promoting the growth or in this context, livestock and poultry production in the abuse and misuse of antibiotics is a common phenomenon, which is a great challenge to human and animal health and disease prevention and control bacteria. Florfenicol (FLO) is a new kind of broad-spectrum antibiotics for Veterinary Clinical College, has become the most widely used animal husbandry and aquaculture in the process, one of the largest amount of antimicrobial drug use. "The endosymbiotic theory" that the mitochondrial origin of eukaryotes from ancient bacteria, archaea parasitic in primitive organisms with long interaction The symbiosis and evolution and evolved into mitochondria, mitochondria ribosomes in the structure and function of the bacterial ribosome has high similarity. Based on this, the eukaryotic cell mitochondrial ribosomes is easily interfered by certain antibiotics, and to animal body or cell toxicity can be combined with the severity of.FLO closely with the bacterial ribosome A sites and inhibit peptidyl transferase activity by affecting protein synthesis and eventually kill bacteria, but also affect the synthesis of eukaryotic mitochondrial proteins. With a large amount of FLO should be used, especially on the toxicity and embryo toxicity hematopoietic immune toxicity reported more and more, but on the basis of FLO on organ function or tissue physiological effects observed or detected, we haven't seen FLO toxicity at the molecular level is clearly F reported. The effect of LO on mitochondrial structure and function, and on cell survival, cell proliferation and cell homeostasis toxicity in this study fibroblasts as the research object, at the cellular and molecular level study on the effect of FLO on the structure and function of mitochondria, and mitochondrial damage on cell proliferation, cell viability and mitochondrial effects autophagy and its mechanism, reveals the specific mechanism of FLO induced into fiber cell toxicity at the molecular level. First, FLO induced mitochondrial mitochondrial structure and function damage in eukaryotic cells as the main place for aerobic respiration, for cellular activity the integrity of its structure and function is very important. This paper focused on FLO effect of mitochondrial structure and function, with different doses of FLO (0.4,0.1,0.025 mg/m L) treated cells after 48 h, mainly for the following experiments: (1) Western blot respectively. The expression level of FLO on the measurement of mitochondrial ribosomal protein Cox encoding I encoding protein and cytoplasmic ribosomes Cox IV; (2) influence the transmission electron microscopy FLO on the number and structure of mitochondria in cells; (3) were detected FLO on ROS mitochondrial membrane potential and intracellular flow cytometry. Surgery (ROS) effect of production; (4) were determined by FLO on mitochondrial respiratory chain complex I biochemical method, influence the production of ATP catalytic activity and cell II and IV. The results show that FLO can significantly inhibit the mitochondrial ribosomal translation of mitochondrial proteins such as Cox and I expression, significantly decreased mitochondrial respiration the chain complex I and IV activity (P0.01), significantly decreased the mitochondrial membrane potential (P0.05 or P0.01) and intracellular levels of ATP (P0.05 or P0.01) and increase the production of ROS; from the structure, at FLO after cell swelling, Vacuolization, cristae disappeared. These results showed that the damage of structure and physiological function of mitochondria was severely affected by FLO. Two, mitochondrial dysfunction induced by FLO inhibition of cell proliferation activity of normal mitochondria plays an important role in the regulation of cell cycle and cell proliferation, development and other aspects, in view of the fact that FLO can cause mitochondrial dysfunction obviously, then we from the following aspects of the FLO effect on cell survival, cell cycle and proliferation regulation: (1) were treated with different doses of FLO cells in 12 h, 24 h, 36 h, 48 h, 60 h and 72 h, the cell count of cell proliferation curve; (2) the determination of different dose of FLO 24 cells h CCK-8 reduction method, 48 h and 72 h on the cell proliferation effect; (3) Ed U incorporation assay with different doses of FLO treatment for 48 h replication activity of cell DNA; (4) different doses of FLO were detected by flow cytometry The distribution of cell cycle; (5) flow cytometry and caspase-3 assay. FLO activity analysis on cell apoptosis; (6) lactate dehydrogenase (LDH) release assay of FLO on cell death rate; (7) the activation of Western blot detection of FLO on cellular proliferation related signaling pathways influence. The results showed that: (1) FLO can lead to dose related reduction of cell viability, reflected in the decline in the absolute number of cells and the proliferation activity decreased (P0.01); (2) Ed U incorporation assay and cell cycle assay showed that FLO can significantly reduce the occurrence of DNA replication and the proportion of cells in the cell cycle of G0/G1 block (P0.05 or P0.01); (3) using D- galactose medium forcing cells only through the mitochondrial respiratory chain of energy supply in order to facilitate the mitochondrial toxicity of substances on mitochondrial toxicity is more obvious, after giving FLO found a variety of cell surface A more serious inhibition of cell viability (P0.01), showed that FLO damage on the mitochondrial respiratory chain and cellviability decreased by direct correlation; (4) FLO on cell apoptosis rate and necrosis rate had no significant effect (P0.05), indicating that FLO induced cell viability decreased only related to cell proliferation is slow, no significant correlation with cell death; (5) from the mechanism, FLO activated AMPK-m TOR-p70S6K pathway, namely FLO activated AMPK (phosphorylated) expression and phosphorylation of M TOR and phosphorylated p70S6K expression, while ROS-p53-p21 pathway was also activated, AMPK specific inhibitor Compound C and antioxidant NAC which can activate specific inhibition of the corresponding pathway and improve the cell proliferation activity was inhibited FLO (P0.05 or P0.01). Three FLO, leading to cell damage within the mitochondria and remove obstacles and senescence occurs on mitochondrial autophagy damage line Critical particle clearance and maintain cellular homeostasis, whereas FLO can cause mitochondrial dysfunction obviously, we then from the following aspects of removing FLO on cell injury of mitochondria that mitochondrial autophagy: (1) Western blot detection of FLO on the intracellular mitochondrial mass (mitochondrial protein Tim23) effect; (2) mitochondrial staining using a mitochondria specific probe Mito Tracker Green, and flow cytometry was used to detect the number of intracellular FLO on mitochondrial effects; (3) specific antibodies using autophagy related protein LC3 staining combined with confocal microscopy formation of FLO cells in autophagy point; (4) expression the level of Western blot FLO on autophagy related protein LC3B II/I and p62, to evaluate the level of autophagy; (5) senescence associated beta galactosidase staining to detect the FLO 8 D treatment caused by the The cumulative effect of mitochondrial damage on cell senescence level; (6) effects of Western blot FLO on the detection of mitochondrial damage to recruit mitochondrial autophagy protein Parkin start key and the level of p62, combined with the ROS scavenger NAC study of p53 protein effect on Parkin translocation to the mitochondria; (7) Mito Tracker Green staining combined with flow cytometry NAC to remove or damage the mitochondrial mitochondrial autophagy function. The results showed that: (1) the function of autophagy is inhibited in FLO treated cells, expressed in autophagy related protein LC3B decreased II and p62 protein expression level increased, no significant difference in the number of intracellular LC3 fluorescence accumulation in different treatment groups (; 2) intracellular mitochondrial membrane protein Tim23 levels increased slightly after FLO administration, Mitotracker Green fluorescent FLO treatment group was significantly increased after FLO treatment showed that mitochondria do not damage Through autophagy effectively cleared, but the accumulation in the cells; (3) Parkin protein from the cytoplasm is recruited to mitochondria is a key link of mitophagy, this study found that after the treatment of FLO mitochondrial Parkin protein and p62 protein recruitment amount did not increase, suggesting mitochondrial translocation and Parkin protein abnormal disorder FLO induced mitochondrial autophagy; (4) using NAC and FLO cells treated with Parkin protein found in mitochondrial disorders can be mitigated, combined with the previous p53 protein Parkin protein inhibiting mitochondrial translocation reports shows that FLO activation of the ROS-p53 pathway and the mitochondrial translocation of Parkin protein inhibits mitochondrial autophagy play inhibitory effect, and then make the injury mitochondria not effectively remove and eventually lead to cell senescence. The research from the molecular level reveals the florfenicol induced fibroblast cell toxicity The specific mechanism, the research results have important significance for the mechanism of toxicity of clear mitochondrial toxicity of drugs, helps to guide the rational application of florfenicol in animal husbandry and aquaculture; at the same time, mitochondria targeted anticancer drug development and a new generation of antibiotics in the mitochondrial safety evaluation has the reference significance.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S859.82

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7 侯澤民;梁曉健;劉白則;;關(guān)于《聚丙烯酰胺水凝膠毒副作用的實驗研究》一文的科學(xué)性的商榷[A];醫(yī)用聚丙烯酰胺水凝膠（奧美定）論文匯編（第二輯）[C];2002年

8 王益平;;正確認識中西藥配伍的不良反應(yīng)及毒副作用[A];浙江省臨床合理用藥和藥劑學(xué)術(shù)研討會論文集[C];2007年

9 張世君;王海霞;王寧;;老年骨腫瘤患者應(yīng)用抗腫瘤藥物毒副作用的監(jiān)護[A];中華護理學(xué)會全國腫瘤護理學(xué)術(shù)交流暨專題講座會議論文匯編[C];2009年

10 楊帆;;中藥產(chǎn)生毒副作用的機理及預(yù)防措施[A];畜牧業(yè)環(huán)境、生態(tài)、安全生產(chǎn)與管理——2010年家畜環(huán)境與生態(tài)學(xué)術(shù)研討會論文集[C];2010年

中國重要報紙全文數(shù)據(jù)庫 前10條

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3 山東農(nóng)業(yè)大學(xué)動物科技學(xué)院 冀貞陽;可相互增強毒副作用的中西藥物[N];江蘇農(nóng)業(yè)科技報;2006年

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中國博士學(xué)位論文全文數(shù)據(jù)庫 前2條

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2 易曉陽;Ⅰ.低溫條件下血小板氧化損傷機制的研究 Ⅱ.血小板冰凍保護劑DMSO的毒副作用初探[D];中國人民解放軍軍事醫(yī)學(xué)科學(xué)院;2017年

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1 盧亮亮;多烯紫杉醇在中晚期老年肺癌患者放射治療中產(chǎn)生的增敏及毒副作用臨床研究[D];山東大學(xué);2016年

2 陶思錚;艾滋病患者HAART相關(guān)臨床毒副作用與線粒體損傷動態(tài)研究[D];昆明醫(yī)科大學(xué);2014年

3 高旭;免疫抑制劑聯(lián)合治療類風(fēng)濕關(guān)節(jié)炎動物模型的毒副作用觀察[D];廣西醫(yī)科大學(xué);2010年

4 曹琦琛;“富士”蘑菇粉抗腫瘤、減輕化療藥毒副作用及毒理學(xué)研究[D];遼寧中醫(yī)藥大學(xué);2007年

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