本文選題：納米毒理學(xué) + 離子選擇性微電極�。� 參考：《東北師范大學(xué)》2015年碩士論文

【摘要】：碳納米管(Carbon Nanotubes,CNTs)是一種重要的納米材料,其獨特的結(jié)構(gòu)和物理化學(xué)性質(zhì),使得它在工業(yè)、能源、環(huán)境、生物醫(yī)藥等領(lǐng)域有著廣闊的應(yīng)用前景。而隨著碳納米材料的廣泛應(yīng)用,動植物和人類與CNTs等碳納米材料接觸的機會也越來越大,對CNTs的生物安全性進(jìn)行研究和評價就顯得十分必要,而且已有研究表明CNTs確實會對生物體產(chǎn)生毒性作用。雖然有關(guān)CNTs的生物安全性研究已經(jīng)取得了一定的進(jìn)展,但對于其產(chǎn)生毒性作用的機理還需要進(jìn)行更加深入的探究。眾所周知,游離的鈣離子作為第二信使在細(xì)胞信號轉(zhuǎn)導(dǎo)過程中起著非常重要的作用,是維持生物體正常生命活動的必須營養(yǎng)元素。研究CNTs對細(xì)胞鈣離子代謝的影響,對于揭示CNTs產(chǎn)生生物毒性的機理,準(zhǔn)確評價CNTs生物安全性具有重要的意義。本文主要采用鈣離子選擇性微電極技術(shù)研究了在多壁碳納米管(Multi-walled Carbon Nanotubes,MWCNTs)作用下蘆薈細(xì)胞原生質(zhì)體的鈣離子釋放問題。鈣離子選擇性微電極技術(shù)可以實現(xiàn)無損傷的原位實時測量,也無需像熒光探針方法那樣對細(xì)胞進(jìn)行紫外照射,因此可以長時間連續(xù)測量。論文首先對鈣離子選擇性微電極的制備過程及其性能進(jìn)行了研究,對于電極拉制溫度、硅烷化溫度和硅烷化試劑用量等制備條件進(jìn)行了優(yōu)化,成功制備了性能優(yōu)良的鈣離子選擇性微電極。經(jīng)測試,電極的轉(zhuǎn)化效率在大于90%,當(dāng)鈣離子濃度在10-5~10-1 mol/L范圍內(nèi)時,電極的響應(yīng)時間小于1 s。在此基礎(chǔ)上,對MWCNTs作用下蘆薈細(xì)胞原生質(zhì)體的鈣離子釋放進(jìn)行了研究。實驗中所用的蘆薈細(xì)胞原生質(zhì)體通過酶解法制取,用FDA法檢測其活性。MWCNTs在使用前進(jìn)行純化處理,XRD測試未發(fā)現(xiàn)催化劑殘留。經(jīng)酶解法提取的原生質(zhì)體先用無鈣培養(yǎng)基進(jìn)行洗滌,使背景鈣離子濃度降至約10-5 mol/L,之后在含有不同濃度和不同直徑MWCNTs的無鈣培養(yǎng)基中進(jìn)行培養(yǎng),并連續(xù)測量原生質(zhì)體釋放到培養(yǎng)基中的鈣離子濃度。結(jié)果發(fā)現(xiàn),與相同條件下培養(yǎng)的對照組對比,MWCNTs的加入加速了原生質(zhì)體鈣離子的釋放,使得培養(yǎng)基中的鈣離子濃度在較短的時間內(nèi)達(dá)到飽和。當(dāng)MWCNTs濃度在20 mg/L~40 mg/L范圍時,隨著MWCNTs濃度的增加,鈣離子釋放速度加快;而當(dāng)MWCNTs的濃度達(dá)到50 mg/L時,鈣離子釋放速度略有降低。對兩種不同直徑的MWCNTs進(jìn)行比較,直徑為20-30 nm的MWCNTs較直徑小于8 nm的MWCNTs對鈣離子釋放的加速作用更為顯著。上述結(jié)果從一個新的層面揭示了MWCNTs對植物細(xì)胞的影響,對全面理解MWCNTs與植物細(xì)胞間的相互作用形式和作用機理,以及對MWCNTs的生物安全性進(jìn)行評價具有重要作用。
[Abstract]:Carbon Nanotubeson (CNTs) is a kind of important nanometer material, its unique structure and physical and chemical properties make it have a broad application prospect in the fields of industry, energy, environment, biomedicine and so on. With the wide application of carbon nanomaterials, it is necessary to study and evaluate the biological safety of CNTs because of the increasing chance of contact between animals and plants and humans and other carbon nanomaterials such as CNTs. And studies have shown that CNTs does have toxic effects on organisms. Although some progress has been made in the study of biosafety of CNTs, the mechanism of its toxic effect needs to be further explored. It is well known that free calcium plays a very important role in cell signal transduction as the second messenger and is the essential nutrient element to maintain the normal life of organism. It is of great significance to study the effect of CNTs on the calcium metabolism of cells and to reveal the mechanism of CNTs biotoxicity and evaluate the biological safety of CNTs accurately. Calcium ion release from the protoplasts of aloe cells under the action of multi-walled Carbon Nanotubesman MWCNTswas studied by calcium ion-selective microelectrode technique. Calcium ion selective microelectrode technique can be used to realize in situ real-time measurement without damage and without ultraviolet irradiation as fluorescence probe method, so it can be measured continuously for a long time. Firstly, the preparation process and properties of calcium ion selective microelectrode were studied. The preparation conditions such as electrode drawing temperature, silanization temperature and amount of silane reagent were optimized. Calcium ion selective microelectrode with excellent properties was successfully prepared. The results show that the conversion efficiency of the electrode is greater than 90 and the response time of the electrode is less than 1 s when the concentration of calcium ion is in the range of 10 ~ (-5) ~ 10 ~ (-1) mol/L. On this basis, calcium release from protoplasts of aloe cells induced by MWCNTs was studied. The protoplasts of aloe cells were prepared by enzymatic hydrolysis. The activity of Aloe cell protoplasts was determined by FDA method. The protoplasts extracted by enzymatic hydrolysis were washed with calcium free medium, and the background calcium ion concentration was reduced to about 10 ~ (-5) mol / L, and then cultured in a calcium free medium containing different concentrations and different diameters of MWCNTs. The calcium concentration released by protoplasts into the medium was measured continuously. The results showed that the addition of MWCNTs accelerated the release of Ca ~ (2 +) from protoplasts compared with the control group under the same culture conditions, and the concentration of Ca ~ (2 +) in the medium reached saturation in a relatively short time. When the concentration of MWCNTs was in the range of 20 mg/L~40 mg/L, the release rate of Ca ~ (2 +) was accelerated with the increase of MWCNTs concentration, while the release rate of Ca ~ (2 +) was slightly decreased when the concentration of MWCNTs reached 50 mg/L. Compared with MWCNTs with different diameters, MWCNTs with diameter of 20-30 nm is more effective than MWCNTs with diameter less than 8 nm in accelerating the release of Ca ~ (2 +). These results reveal the effect of MWCNTs on plant cells from a new perspective, and play an important role in understanding the interaction between MWCNTs and plant cells and in evaluating the biological safety of MWCNTs.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：TB383.1

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