體外研究羧基化多壁碳納米管誘發(fā)的氧化應(yīng)激效應(yīng)與機(jī)理
發(fā)布時(shí)間：2018-10-16 11:35
【摘要】：自從1991年碳納米管發(fā)現(xiàn)以來,由于其具有奇特的電學(xué)性質(zhì)、光學(xué)和光電子性質(zhì)、力學(xué)和機(jī)電性質(zhì)、磁學(xué)和電磁性質(zhì)以及熱學(xué)和熱電性質(zhì),因而在諸多領(lǐng)域都顯現(xiàn)出廣闊的應(yīng)用潛力。與此同時(shí),碳納米管的毒性作用也備受關(guān)注。碳納米管通常分為單壁碳納米管和多壁碳納米管。由于多壁碳納米管具有較強(qiáng)的生物相容性,進(jìn)而,多壁碳納米管(MWCNTs)的安全性受到了更多的關(guān)注。因此,本論文選用分散性較好的羧基化多壁碳納米管(MWCNTs-COOH)展開研究。有研究指出,納米材料進(jìn)入生物體產(chǎn)生的毒性作用,與誘發(fā)氧化應(yīng)激效應(yīng)相關(guān)。氧化應(yīng)激效應(yīng)將會(huì)導(dǎo)致機(jī)體內(nèi)活性氧(ROS)的大量產(chǎn)生�；钚匝�(ROS)在體內(nèi)積聚達(dá)到一定的量,機(jī)體將產(chǎn)生氧化應(yīng)激損傷,進(jìn)而顯現(xiàn)出各類病癥。因此,通過研究碳納米管誘發(fā)機(jī)體產(chǎn)生的氧化應(yīng)激效應(yīng)與機(jī)理,將有助于進(jìn)一步深入研究和認(rèn)識碳納米管生物毒性的作用機(jī)理,為今后對碳納米管毒性作用的研究提供更多的科學(xué)依據(jù)。本論文將結(jié)合分子水平和細(xì)胞水平,從體外研究羧基化多壁碳納米管誘發(fā)的氧化應(yīng)激效應(yīng)與機(jī)理。研究的主要內(nèi)容如下：首先,通過紅外光譜和高分辨透射電鏡對MWCNTs-COOH進(jìn)行了表征。其次,通過多種光譜學(xué)的方法和等溫滴定量熱法等手段,研究了MWCNTs-COOH結(jié)果表明,MWCNTs-COOH與兩種酶的相互作用都比較弱,且都是以疏水作用力為主的弱吸熱反應(yīng)。MWCNTs-COOH對兩種酶的結(jié)構(gòu)均沒有產(chǎn)生明顯的影響。酶活性實(shí)驗(yàn)表明,MWCNTs-COOH未對它們的生理功能產(chǎn)生明顯的影響。最后,利用Muse智能觸控細(xì)胞分析儀,研究了分子水平濃度下MWCNTs-COOH對原代小鼠肝細(xì)胞成活率的影響。以此為基礎(chǔ),測定了MWCNTs-COOH對細(xì)胞內(nèi)過氧化酶、超氧化物歧化酶活性和丙二醛(MDA)含量的影響,探討了MWCNTs-COOH誘發(fā)細(xì)胞產(chǎn)生的氧化應(yīng)激效應(yīng)。主要結(jié)論：①WCNTs-COOH對細(xì)胞活力呈時(shí)間依賴效應(yīng)；②CAI、SOD的活性都受到了抑制,MWCNTs-COOH誘發(fā)肝細(xì)胞產(chǎn)生氧化應(yīng)激；③MDA的含量發(fā)生變化,但沒有顯著性差異,說明MWCNTs-COOH未對細(xì)胞造成明顯的氧化應(yīng)激損傷。通過對比兩種不同研究手段的實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)：在相同的MWCNTs-COOH濃度下,分子和細(xì)胞水平上抗氧化應(yīng)激關(guān)鍵酶活性的變化不一致,揭示出MWCNTs-COOH不是直接作用于抗氧化應(yīng)激關(guān)鍵酶,通過影響其結(jié)構(gòu)而改變其生理活性；而是通過誘發(fā)氧化應(yīng)激效應(yīng),間接影響抗氧化應(yīng)激關(guān)鍵酶的生理活性。細(xì)胞內(nèi)抗氧化應(yīng)激關(guān)鍵酶的活性與氧化應(yīng)激效應(yīng)有著復(fù)雜的關(guān)聯(lián),這將為今后深入研究碳納米管的生物毒性作用提供更多的參考依據(jù)。
[Abstract]:Since the discovery of carbon nanotubes in 1991, due to their unique electrical, optical and optoelectronic properties, mechanical and mechanical properties, magnetic and electromagnetic properties, and thermal and thermoelectric properties, As a result, there are broad application potential in many fields. At the same time, the toxicity of carbon nanotubes (CNTs) has attracted much attention. Carbon nanotubes are usually divided into single-walled carbon nanotubes and multi-walled carbon nanotubes. The safety of multiwalled carbon nanotubes (MwCNTs) has attracted more and more attention due to their strong biocompatibility. Therefore, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) with good dispersity were selected in this thesis. Some studies have indicated that the toxic effect of nanomaterials entering organism is related to the effect of induced oxidative stress. Oxidative stress will lead to the production of reactive oxygen species (ROS) in the body. Reactive oxygen species (Ros) (ROS) accumulates to a certain amount in the body, which will lead to oxidative stress damage, and then show various diseases. Therefore, by studying the oxidative stress effect and mechanism induced by carbon nanotubes, it will be helpful to further study and understand the mechanism of biological toxicity of carbon nanotubes. It provides more scientific basis for the study of the toxicity of carbon nanotubes in the future. In this paper, the oxidative stress induced by carboxylated multiwalled carbon nanotubes (MMWNTs) was studied in vitro at the molecular and cellular levels. The main contents are as follows: firstly, MWCNTs-COOH was characterized by infrared spectroscopy and high resolution transmission electron microscope. Secondly, by means of various spectroscopic methods and isothermal drop quantitative calorimetry, the MWCNTs-COOH results show that the interaction between MWCNTs-COOH and the two enzymes is weak. Both of them were weak endothermic reactions dominated by hydrophobic forces. MWCNTs-COOH had no obvious effect on the structure of the two enzymes. The enzyme activity experiment showed that MWCNTs-COOH had no obvious effect on their physiological function. Finally, the effect of MWCNTs-COOH on the survival rate of primary mouse hepatocytes at molecular level was studied by Muse intelligent touch cell analyzer. On this basis, the effects of MWCNTs-COOH on the activities of peroxidase, superoxide dismutase and malondialdehyde (MDA) in cells were determined, and the oxidative stress induced by MWCNTs-COOH was discussed. Conclusion: 1WCNTs-COOH has a time-dependent effect on cell viability, the activity of 1WCNTs-COOH is inhibited, MWCNTs-COOH induces oxidative stress in hepatocytes, the content of 3MDA changes, but there is no significant difference. The results showed that MWCNTs-COOH did not cause obvious oxidative stress damage to cells. By comparing the results of two different research methods, it was found that at the same concentration of MWCNTs-COOH, the changes of the key enzymes of antioxidant stress at the molecular and cellular levels were not consistent, which revealed that MWCNTs-COOH did not act directly on the key enzymes of antioxidant stress. The physiological activity was changed by affecting its structure, and the physiological activity of key enzymes of antioxidant stress was indirectly affected by inducing oxidative stress effect. The activities of the key enzymes of intracellular antioxidant stress are closely related to the oxidative stress effect, which will provide more references for the further study of the biological toxicity of carbon nanotubes in the future.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：O613.71;TB383.1
本文編號：2274234
[Abstract]:Since the discovery of carbon nanotubes in 1991, due to their unique electrical, optical and optoelectronic properties, mechanical and mechanical properties, magnetic and electromagnetic properties, and thermal and thermoelectric properties, As a result, there are broad application potential in many fields. At the same time, the toxicity of carbon nanotubes (CNTs) has attracted much attention. Carbon nanotubes are usually divided into single-walled carbon nanotubes and multi-walled carbon nanotubes. The safety of multiwalled carbon nanotubes (MwCNTs) has attracted more and more attention due to their strong biocompatibility. Therefore, carboxylated multi-walled carbon nanotubes (MWCNTs-COOH) with good dispersity were selected in this thesis. Some studies have indicated that the toxic effect of nanomaterials entering organism is related to the effect of induced oxidative stress. Oxidative stress will lead to the production of reactive oxygen species (ROS) in the body. Reactive oxygen species (Ros) (ROS) accumulates to a certain amount in the body, which will lead to oxidative stress damage, and then show various diseases. Therefore, by studying the oxidative stress effect and mechanism induced by carbon nanotubes, it will be helpful to further study and understand the mechanism of biological toxicity of carbon nanotubes. It provides more scientific basis for the study of the toxicity of carbon nanotubes in the future. In this paper, the oxidative stress induced by carboxylated multiwalled carbon nanotubes (MMWNTs) was studied in vitro at the molecular and cellular levels. The main contents are as follows: firstly, MWCNTs-COOH was characterized by infrared spectroscopy and high resolution transmission electron microscope. Secondly, by means of various spectroscopic methods and isothermal drop quantitative calorimetry, the MWCNTs-COOH results show that the interaction between MWCNTs-COOH and the two enzymes is weak. Both of them were weak endothermic reactions dominated by hydrophobic forces. MWCNTs-COOH had no obvious effect on the structure of the two enzymes. The enzyme activity experiment showed that MWCNTs-COOH had no obvious effect on their physiological function. Finally, the effect of MWCNTs-COOH on the survival rate of primary mouse hepatocytes at molecular level was studied by Muse intelligent touch cell analyzer. On this basis, the effects of MWCNTs-COOH on the activities of peroxidase, superoxide dismutase and malondialdehyde (MDA) in cells were determined, and the oxidative stress induced by MWCNTs-COOH was discussed. Conclusion: 1WCNTs-COOH has a time-dependent effect on cell viability, the activity of 1WCNTs-COOH is inhibited, MWCNTs-COOH induces oxidative stress in hepatocytes, the content of 3MDA changes, but there is no significant difference. The results showed that MWCNTs-COOH did not cause obvious oxidative stress damage to cells. By comparing the results of two different research methods, it was found that at the same concentration of MWCNTs-COOH, the changes of the key enzymes of antioxidant stress at the molecular and cellular levels were not consistent, which revealed that MWCNTs-COOH did not act directly on the key enzymes of antioxidant stress. The physiological activity was changed by affecting its structure, and the physiological activity of key enzymes of antioxidant stress was indirectly affected by inducing oxidative stress effect. The activities of the key enzymes of intracellular antioxidant stress are closely related to the oxidative stress effect, which will provide more references for the further study of the biological toxicity of carbon nanotubes in the future.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：O613.71;TB383.1
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