【摘要】：納米金棒(Gold nanorods,AuNRs)是指尺度介于1-100nm之間的棒狀納米金顆粒,在光學(xué)特性、量子隧道效應(yīng)、生物相容性、表面易修飾性和穩(wěn)定性等方面具有獨(dú)特的物理及化學(xué)屬性,因此被認(rèn)為是最具應(yīng)用前景的納米材料之一。近年來,隨著納米技術(shù)在生物醫(yī)學(xué)領(lǐng)域的發(fā)展,AuNRs在細(xì)胞和動(dòng)物活體成像、藥物和基因傳遞以及多種疾病的診斷和治療等方面的運(yùn)用越來越多。然而,隨著AuNRs廣泛應(yīng)用,人們接觸AuNRs的機(jī)會(huì)日益增多,AuNRs暴露所引起的生物安全問題也受到越來越多的關(guān)注。因此,探究AuNRs對(duì)生物體可能潛在的影響及其機(jī)制是納米金材料得到更加安全、廣泛運(yùn)用的基礎(chǔ)。本研究擬首先采用不同濃度AuNRs處理A549細(xì)胞6h、12h和24h,然后通過CCK-8、LDH實(shí)驗(yàn)觀察AuNRs對(duì)A549細(xì)胞活力、細(xì)胞膜損傷的影響。在此基礎(chǔ)上,運(yùn)用透射電鏡觀察AuNRs對(duì)細(xì)胞超微結(jié)構(gòu)的影響,并初步判斷AuNRs的細(xì)胞毒性。進(jìn)一步,擬運(yùn)用Western blot技術(shù)、激光掃描共聚焦顯微技術(shù)和其他分子生物學(xué)方法來觀察AuNRs細(xì)胞毒性是否與其通過損傷線粒體相關(guān)功能、促進(jìn)氧化應(yīng)激進(jìn)而誘導(dǎo)細(xì)胞自噬有關(guān),相關(guān)實(shí)驗(yàn)結(jié)果如下:一、AuNRs對(duì)A549細(xì)胞毒性的影響首先,將正常培養(yǎng)的A549細(xì)胞給予不同濃度AuNRs處理(0μg/ml,0.5μg/ml,1μg/ml,2μg/ml,4μg/ml),分別處理6h、12h、24h后觀察其對(duì)細(xì)胞活力、細(xì)胞膜損傷和細(xì)胞超微結(jié)構(gòu)的影響,實(shí)驗(yàn)結(jié)果如下:1.AuNRs對(duì)A549細(xì)胞活力的影響CCK-8實(shí)驗(yàn)結(jié)果表明,采用不同濃度AuNRs處理后,A549細(xì)胞活力呈劑量、時(shí)間依賴性降低。提示,AuNRs細(xì)胞毒性呈劑量、時(shí)間依賴性增加。2.AuNRs對(duì)A549細(xì)胞膜損傷的影響LDH實(shí)驗(yàn)結(jié)果表明,采用不同濃度AuNRs處理后,A549細(xì)胞呈現(xiàn)LDH滲漏且上清LDH呈劑量、時(shí)間依賴性增加。提示,AuNRs細(xì)胞毒性呈劑量、時(shí)間依賴性增加,AuNRs作用的濃度越高、時(shí)間越長(zhǎng),對(duì)細(xì)胞的毒性越大。3.AuNRs對(duì)A549細(xì)胞超微結(jié)構(gòu)的影響透射電鏡研究結(jié)果表明,AuNRs能夠進(jìn)入A549細(xì)胞,并以單個(gè)顆�；蚓奂w的形式存在于細(xì)胞質(zhì)和部分膜囊泡中,而細(xì)胞核未觀察到AuNRs。此外,細(xì)胞中部分線粒體呈不同程度腫脹,并且自噬小體數(shù)目較對(duì)照組顯著增加。二、aunrs對(duì)a549細(xì)胞自噬的影響研究發(fā)現(xiàn),自噬是納米材料細(xì)胞毒性的重要機(jī)制之一。那么aunrs對(duì)a549細(xì)胞毒性是否與自噬有關(guān)呢?我們進(jìn)行以下研究,實(shí)驗(yàn)結(jié)果如下:1.aunrs對(duì)a549細(xì)胞自噬標(biāo)志蛋白lc3的影響激光掃描共聚焦顯微鏡觀察結(jié)果表明,不同濃度aunrs處理a549細(xì)胞6h后,lc3表達(dá)呈劑量依賴性增加。其中,con組lc3主要表達(dá)并集中于細(xì)胞核,而隨著aunrs濃度的增加,lc3表達(dá)逐漸從細(xì)胞核轉(zhuǎn)移到細(xì)胞質(zhì),細(xì)胞核lc3表達(dá)逐步減少并呈空泡化。再將a549細(xì)胞給予2μg/ml的aunrs分別處理6h、12h和24h并觀察lc3表達(dá)情況,結(jié)果表明,a549細(xì)胞lc3表達(dá)呈時(shí)間依賴性增加。con組lc3主要表達(dá)并集中于細(xì)胞核,而隨著aunrs暴露時(shí)間的增加,lc3表達(dá)逐漸從細(xì)胞核轉(zhuǎn)移到細(xì)胞質(zhì),細(xì)胞核lc3表達(dá)逐步減少并呈空泡化。2.aunrs對(duì)a549細(xì)胞自噬相關(guān)蛋白的影響westernblot結(jié)果表明,不同濃度aunrs處理a549細(xì)胞6h后,lc3-ii、atg4、atg16和beclin1蛋白表達(dá)顯著增加,而自噬底物蛋白p62表達(dá)水平顯著降低。提示,aunrs誘導(dǎo)a549細(xì)胞發(fā)生自噬呈劑量、時(shí)間依賴性增加。3.cq能夠抑制aunrs誘導(dǎo)的自噬westernblot結(jié)果表明,正常培養(yǎng)條件下con組atg16、lc3-ii少量表達(dá),給予cq處理后atg16、lc3-ii表達(dá)較con組顯著增加,表明cq可以抑制基礎(chǔ)水平自噬溶酶體降解。給予aunrs處理后,atg16、lc-ii表達(dá)較對(duì)照組顯著增加。aunrs和cq共同處理a549細(xì)胞6h后,atg16高表達(dá)可被cq逆轉(zhuǎn),而lc3-ii表達(dá)則進(jìn)一步增加。這一結(jié)果表明,cq不僅可以抑制由aunrs誘導(dǎo)自噬的起始,而且可以抑制自噬溶酶體降解。三、氧化應(yīng)激介導(dǎo)aunrs誘導(dǎo)a549細(xì)胞產(chǎn)生的自噬近年來有研究發(fā)現(xiàn),氧化應(yīng)激是誘導(dǎo)細(xì)胞自噬的重要原因。那么aunrs誘導(dǎo)a549細(xì)胞發(fā)生自噬是否與氧化應(yīng)激有關(guān)?我們的實(shí)驗(yàn)結(jié)果如下:1.抗氧化劑mntbap和nac能夠抑制aunrs誘導(dǎo)的自噬westernblot結(jié)果表明,con組中l(wèi)c3-ii表達(dá)水平較低,mntbap、nac處理后其表達(dá)水平輕微降低但無統(tǒng)計(jì)學(xué)意義。而aunrs處理6h后,lc3-ii表達(dá)顯著增加,并且這一效應(yīng)能被mntbap、nac所逆轉(zhuǎn)。提示,mntbap、nac可以改善aunrs誘導(dǎo)a549細(xì)胞產(chǎn)生的自噬。激光掃描共聚焦顯微鏡研究表明,con組、nac組中l(wèi)c3表達(dá)較弱且主要集中于細(xì)胞核,表明其自噬水平很低。而aunrs處理6h后,lc3蛋白質(zhì)從細(xì)胞核轉(zhuǎn)移到細(xì)胞質(zhì),細(xì)胞核lc3表達(dá)逐步減少并呈空泡化。而抗氧化劑nac能夠逆轉(zhuǎn)上述效應(yīng)。2.抗氧化劑MnTBAP和NAC能夠逆轉(zhuǎn)AuNRs誘導(dǎo)的ROS增加激光掃描共焦顯微鏡研究表明,不同濃度AuNRs處理A549細(xì)胞6h后,Con組未觀察到ROS陽性細(xì)胞,表明其ROS水平很低。而隨著AuNRs濃度的增加,陽性ROS細(xì)胞數(shù)量和綠色熒光亮度明顯增加且呈劑量依賴性,表明AuNRs能夠誘導(dǎo)ROS產(chǎn)生,濃度越高,ROS水平越高。而抗氧化劑Mn TBAP、NAC能夠逆轉(zhuǎn)上述效應(yīng)。3.抗氧化劑MnTBAP和NAC能夠改善AuNRs誘導(dǎo)細(xì)胞氧化應(yīng)激狀態(tài)T-AOC、GSH/GSSG檢測(cè)結(jié)果表明,AuNRs處理組T-AOC、GSH/GSSG較Con組顯著降低,表明AuNRs處理后細(xì)胞抗氧化能力顯著降低,而MnTBAP、NAC處理6h后能夠逆轉(zhuǎn)上述效應(yīng)。4.抗氧化劑NAC能夠逆轉(zhuǎn)AuNRs誘導(dǎo)線粒體功能的降低線粒體膜電位和ATP含量測(cè)定實(shí)驗(yàn)結(jié)果表明,AuNRs處理后其線粒體膜電位和ATP含量較Con組顯著降低,表明AuNRs處理細(xì)胞后其線粒體功能降低,而NAC處理6h后能夠逆轉(zhuǎn)上述效應(yīng)。Western blot結(jié)果表明,AuNRs處理組UCP2表達(dá)顯著降低。NAC處理6h后UCP2蛋白表達(dá)顯著增加,提示UCP2蛋白表達(dá)降低與線粒體功能改變有關(guān)。結(jié)論:1.AuNRs能夠進(jìn)入A549細(xì)胞,引起細(xì)胞超微結(jié)構(gòu)改變。AuNRs具有一定的細(xì)胞毒性且呈劑量、時(shí)間依賴性增加,AuNRs濃度越高、作用時(shí)間越長(zhǎng),細(xì)胞毒性越大。2.AuNRs能夠誘導(dǎo)A549細(xì)胞產(chǎn)生自噬并能被自噬抑制劑CQ所抑制,自噬標(biāo)志蛋白LC3、自噬相關(guān)蛋白ATG16、ATG4、Beclin-1表達(dá)增加,而自噬底物蛋白P62表達(dá)降低且呈劑量依賴性。3.AuNRs能夠影響A549細(xì)胞線粒體相關(guān)功能,進(jìn)而導(dǎo)致細(xì)胞抗氧化應(yīng)激能力降低、ROS水平增加,后者介導(dǎo)了AuNRs誘導(dǎo)A549細(xì)胞產(chǎn)生的自噬。
[Abstract]:Gold nanorods (AuNRs) are rod-like gold nanoparticles with a scale of 1-100 nm. They have unique physical and chemical properties in optical properties, quantum tunneling effect, biocompatibility, surface modification and stability, so they are considered as one of the most promising nanomaterials in recent years. With the development of biomedical technology, AuNRs are used more and more in cell and animal imaging, drug and gene delivery, diagnosis and treatment of various diseases. However, with the wide application of AuNRs, people have more and more opportunities to contact AuNRs, and the biological safety problems caused by AuNRs exposure have attracted more and more attention. Therefore, to explore the potential effect of AuNRs on organisms and its mechanism is the basis for the safety and widespread use of AuNRs. In this study, different concentrations of AuNRs were used to treat A549 cells for 6 h, 12 h and 24 h, and then CCK-8 and LDH were used to observe the effect of AuNRs on A549 cell viability and membrane damage. The effect of AuNRs on the ultrastructure of cells was observed by transmission electron microscopy, and the cytotoxicity of AuNRs was preliminarily judged. Furthermore, Western blot, laser scanning confocal microscopy and other molecular biology methods were used to observe whether the cytotoxicity of AuNRs was related to mitochondrial damage, promoting oxidative stress and inducing cells. The results were as follows: 1. The effects of AuNRs on cytotoxicity of A549 cells were studied. Firstly, the A549 cells were treated with different concentrations of AuNRs (0 ug/ml, 0.5 ug/ml, 1 ug/ml, 2 ug/ml, 4 ug/ml) for 6 hours, 12 hours and 24 hours respectively. The results were as follows: 1. The results of CCK-8 showed that the activity of A549 cells decreased in a dose-and time-dependent manner after treatment with different concentrations of AuNRs, suggesting that the cytotoxicity of AuNRs increased in a dose-and time-dependent manner. It was suggested that the cytotoxicity of AuNRs increased in a dose-and time-dependent manner. The higher the concentration of AuNRs, the longer the time, the greater the cytotoxicity. 3. The transmission electron microscopic study of the effect of AuNRs on the ultrastructure of A549 cells showed that AuNRs could enter A549 cells in a single particle. In addition, some mitochondria were swollen in different degrees and the number of autophagosomes was significantly increased compared with the control group. Second, the effect of AuNRs on autophagy of A549 cells was found to be one of the important mechanisms of cytotoxicity of nanomaterials. The results were as follows: 1. The effect of AuNRs on autophagy marker protein LC3 of A549 cells was observed by laser scanning confocal microscopy. The results showed that the expression of LC3 increased in a dose-dependent manner after AuNRs treatment for 6 hours. With the increase of AuNRs concentration, the expression of LC3 was gradually transferred from nucleus to cytoplasm, and the expression of LC3 in nucleus was gradually reduced and vacuolated. Then A549 cells were treated with 2 ug/ml AuNRs for 6 h, 12 h and 24 h respectively. The expression of LC3 in A549 cells increased in a time-dependent manner. The expression of LC3 gradually shifted from nucleus to cytoplasm with the increase of AuNRs exposure time. the expression of LC3 gradually decreased and vacuolated in nucleus. 2. the effect of AuNRs on autophagy-related proteins in A549 cells was studied by Western blot. the results showed that the expression of lc3-ii, atg4, atg16 and Beclin1 eggs in A549 cells treated with different concentrations of AuNRs for 6 hours. CQ could inhibit the expression of atg16 and LC3-II in con group. the expression of atg16 and LC3-II in con group was lower than that in con group. The expression of atg16 and lc-ii increased significantly after AuNRs treatment compared with the control group. After AuNRs and CQ treatment for 6 h, the high expression of atg16 could be reversed by cq, while the expression of LC3-II increased further. These results showed that CQ could not only inhibit the initiation of autophagy induced by aunrs. Oxidative stress mediated autophagy of A549 cells in recent years has found that oxidative stress is an important reason for inducing autophagy. then whether the autophagy of A549 cells induced by AuNRs is related to oxidative stress? Our experimental results are as follows: 1. antioxidant mntbap and NAC energy Inhibition of autophagy induced by AuNRs by Western blot showed that the expression of LC3-II was low in con group, but slightly decreased after treatment with mntbap and nac, but there was no significant difference. after treatment with AuNRs for 6 hours, the expression of LC3-II increased significantly, and this effect could be reversed by mntbap and nac. it suggested that mntbap and NAC could improve the production of A549 cells induced by aunrs. Confocal laser scanning microscopy showed that the expression of LC3 in con group and NAC group was weak and mainly concentrated in the nucleus, indicating that the level of autophagy was very low. Antioxidant MnTBAP and NAC could reverse the augmentation of ROS induced by AuNRs. Laser scanning confocal microscopy showed that after 6 hours of AuNRs treatment, no ROS-positive cells were observed in Con group, suggesting that the ROS level was very low. However, with the increase of AuNRs concentration, the number of ROS-positive cells and green fluorescence intensity increased significantly and was dose-dependent. Antioxidants MnTBAP and NAC could improve T-AOC of AuNRs-induced oxidative stress cells. GSH/GSSG test results showed that T-AOC and GSH/GSSG of AuNRs-treated cells were significantly lower than those of Con-treated cells. The results showed that the mitochondrial membrane potential and ATP content decreased significantly after AuNRs treatment, indicating that the mitochondrial mitochondrial membrane potential and ATP content decreased significantly after AuNRs treatment. The results of Western blot showed that the expression of UCP2 was significantly decreased in AuNRs treated group. The expression of UCP2 was significantly increased after 6 hours of NAC treatment, suggesting that the decrease of UCP2 protein expression was related to the change of mitochondrial function. Conclusion: 1. AuNRs could enter A549 cells and cause ultrastructural changes. The higher the concentration of AuNRs and the longer the duration of action, the greater the cytotoxicity. 2. AuNRs can induce autophagy in A549 cells and can be inhibited by autophagy inhibitor CQ. The expression of autophagy marker protein LC3, autophagy associated protein ATG16, ATG4 and Beclin-1 is increased, while the expression of autophagy substrate protein P62 is increased. AuNRs can affect the mitochondrial function of A549 cells in a dose-dependent manner, which leads to the decrease of antioxidant stress and the increase of ROS level, which mediates the autophagy of A549 cells induced by AuNRs.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R318.08

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本文編號(hào)：2230997