【摘要】：隨著經(jīng)濟(jì)的迅速發(fā)展,人類的醫(yī)療衛(wèi)生條件不斷改善,多達(dá)3000種藥物被用于醫(yī)療,并在環(huán)境中積累。由于污水處理廠的設(shè)備并不能將這些有機(jī)藥物完全去除,因此這些有機(jī)藥物被排放到水環(huán)境中,不斷滲透到地表水、地下水和沉積物中,使其在環(huán)境中的分布具有全球普遍性。其中,雙氯芬酸是一種最常用的、療效最好的非甾類鎮(zhèn)痛消炎藥(NSAIDs),被廣泛用于臨床治療中。由于雙氯芬酸藥品的療效好、用量大,被無節(jié)制地排放到環(huán)境中,并危及到了生態(tài)系統(tǒng)的穩(wěn)定性�，F(xiàn)在我們已知,雙氯芬酸能嚴(yán)重影響植物的生長發(fā)育,但是其抑制植物生長的作用機(jī)制至今尚不清楚。為此,本研究利用煙草BY-2細(xì)胞為材料,探討雙氯芬酸對煙草BY-2細(xì)胞的呼吸代謝及其線粒體功能的影響,利用Oxytherm氧電極(Hansatech,英國)測定不同濃度的雙氯芬酸鈉即時(shí)處理的煙草BY-2細(xì)胞的總呼吸,測定不同處理時(shí)間的煙草BY-2細(xì)胞的總呼吸、細(xì)胞色素途徑(COX)和交替氧化酶(AOX)途徑呼吸以及線粒體的狀態(tài)呼吸,測定不同呼吸途徑抑制劑處理的細(xì)胞呼吸,并利用稱量細(xì)胞干重的方法更直觀的驗(yàn)證雙氯芬酸鈉對煙草BY-2細(xì)胞生長的抑制。進(jìn)而,闡明雙氯芬酸抑制煙草BY-2細(xì)胞生長的作用機(jī)理,為闡明雙氯芬酸抑制植物生長的機(jī)理提供理論依據(jù)。本研究結(jié)果表明,0.2 mmol·L-1雙氯芬酸處理24h后,就顯著抑制了煙草BY-2細(xì)胞的生長,引起細(xì)胞內(nèi)活性氧(ROS)的爆發(fā)和積累,并導(dǎo)致煙草BY-2細(xì)胞死亡。并且,研究發(fā)現(xiàn)一定濃度的雙氯芬酸對煙草BY-2細(xì)胞的呼吸有即時(shí)的抑制作用,且隨著雙氯芬酸的濃度增加,對細(xì)胞呼吸作用的抑制程度也增加,當(dāng)雙氯芬酸的濃度達(dá)到0.2mmol·L-1時(shí),它對煙草BY-2細(xì)胞呼吸作用的抑制程度達(dá)到最大。此外,在雙氯芬酸對煙草BY-2細(xì)胞離體線粒體的直接作用的研究中,我們發(fā)現(xiàn)雙氯芬酸通過抑制細(xì)胞線粒體呼吸電子傳遞鏈上復(fù)合體II、III和IV的活性,導(dǎo)致線粒體中細(xì)胞色素氧化酶(COX)和交替氧化酶(AOX)參與的兩條呼吸電子傳遞途徑均受到不同程度的抑制作用,進(jìn)而反饋抑制了細(xì)胞的糖酵解(EMP)、三羧酸循環(huán)(TCA)和戊糖磷酸途徑(PPP)三個(gè)碳代謝途徑。并且,雙氯芬酸對線粒體I、III、IV三個(gè)狀態(tài)的呼吸均有抑制作用,導(dǎo)致呼吸控制率(RCR)降低。而且,雙氯芬酸也能導(dǎo)致線粒體發(fā)生腫脹,并能降低線粒體膜磷脂的聚集程度,破壞了線粒體的完整性,導(dǎo)致跨膜質(zhì)子梯度形成受阻,進(jìn)而造成ATP合成受抑。因此,我們認(rèn)為煙草BY-2細(xì)胞中經(jīng)過線粒體呼吸電子傳遞鏈上復(fù)合體III和IV的電子傳遞被抑制是雙氯芬酸抑制細(xì)胞呼吸的一個(gè)主要原因。本研究證明了雙氯芬酸對線粒體呼吸鏈上復(fù)合體II,III和IV活性的抑制是它抑制細(xì)胞呼吸的主要原因,這就導(dǎo)致了線粒體代謝發(fā)生紊亂。線粒體能量代謝和物質(zhì)代謝的紊亂進(jìn)一步造成細(xì)胞內(nèi)活性氧(ROS)爆發(fā)和積累,這些都是雙氯芬酸抑制煙草BY-2細(xì)胞生長、導(dǎo)致細(xì)胞死亡的重要原因。
[Abstract]:With the rapid development of the economy, human health conditions have been improving, as many as 3,000 drugs have been used for medical treatment and are accumulated in the environment. Since the equipment of the sewage treatment plant cannot completely remove these organic drugs, these organic drugs are discharged into the water environment and continuously penetrate into the surface water, the ground water and the sediment, so that the distribution of the organic medicine in the environment is universal. Among them, diclofenac is one of the most commonly used non-opioid analgesic and anti-inflammatory drugs (NSAIDs), which are widely used in clinical treatment. Due to the good curative effect of the diclofenac medicine, the dosage is large, and the diclofenac medicine is discharged to the environment without control, and the stability of the ecological system is endangered. It is now known that diclofenac can severely affect the growth and development of plants, but the mechanism to inhibit plant growth is not yet clear. To this end, the effects of diclofenac on the respiratory metabolism and the mitochondrial function of the tobacco by-2 cells were investigated by using the tobacco BY-2 cells, and the total respiration of the tobacco by-2 cells treated with diclofenac sodium at different concentrations was determined by using the Oxytherm oxygen electrode (Hansatch, UK). the total respiration, the cytochrome pathway (COX) and the alternating oxidase (AOX) pathway of the tobacco by-2 cells and the state respiration of the mitochondria were measured for different treatment times, and the cellular respiration of the different respiratory pathway inhibitor treatment was determined, And the inhibition of the growth of the tobacco by-2 cells by the diclofenac sodium is more visually verified by the method for weighing the dry weight of the cells. In addition, the mechanism of diclofenac to inhibit the growth of the tobacco by-2 cells is explained, and the theoretical basis for elucidating the mechanism of diclofenac to inhibit the growth of the plants is provided. The results of this study show that after 24 h of 0.2 mmol 路 L-1 diclofenac acid treatment, the growth of the tobacco by-2 cells is significantly inhibited, and the occurrence and accumulation of reactive oxygen (ROS) in the cells is induced, and the tobacco by-2 cells are caused to die. In addition, it was found that the concentration of diclofenac has an immediate effect on the respiration of the tobacco by-2 cells, and as the concentration of diclofenac increases, the degree of inhibition of cellular respiration is also increased, and when the concentration of the diclofenac is 0.2 mmol 路 L-1, It has the greatest inhibitory effect on the respiration of the tobacco by-2 cells. In addition, in the study of the direct effect of diclofenac on the in vitro mitochondria of the tobacco by-2 cells, we found that diclofenac has been used to inhibit the activity of the complexes II, III and IV on the mitochondrial respiratory electron transfer chain of the cell, The two respiratory electron transport pathways involved in the participation of the cytochrome oxidase (COX) and the alternating oxidase (AOX) in the mitochondria are inhibited by different degrees, and the glycolysis (EMP) of the cells is further suppressed, The three pathways of carbon metabolism in the three-acid cycle (TCA) and the pentose phosphate pathway (PPP). In addition, diclofenac has an inhibitory effect on the respiration of the three states of the mitochondria I, III and IV, leading to a reduction in the respiratory control rate (RCR). In addition, diclofenac can also lead to the swelling of the mitochondria, and the aggregation of the mitochondrial membrane phospholipids can be reduced, the integrity of the mitochondria is destroyed, the formation of the transmembrane proton gradient is blocked, and the ATP synthesis is inhibited. Therefore, we believe that the inhibition of the electron transfer of complexes III and IV through the mitochondrial respiratory electron transfer chain in the tobacco BY-2 cells is a major cause of diclofenac to inhibit cellular respiration. This study has shown that the inhibition of the activity of diclofenac on the complex II, III and IV of the mitochondrial respiratory chain is the main cause of the inhibition of cellular respiration, which leads to a disorder in the metabolism of the mitochondria. The disorder of mitochondrial energy metabolism and substance metabolism further causes the explosion and accumulation of reactive oxygen (ROS) in the cells, which are important reasons for diclofenac to inhibit the growth of the tobacco by-2 cells and lead to cell death.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：X503.231

【共引文獻(xiàn)】

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10 馬s，

本文編號(hào)：2449219