离子选择性微电极制备及碳纳米管作用下芦荟细胞原生质体钙离子释放研究

发布时间：2018-06-06 14:07

本文选题：纳米毒理学 + 离子选择性微电极　；参考：《东北师范大学》2015年硕士论文

【摘要】：碳纳米管(Carbon Nanotubes,CNTs)是一种重要的纳米材料,其独特的结构和物理化学性质,使得它在工业、能源、环境、生物医药等领域有着广阔的应用前景。而随着碳纳米材料的广泛应用,动植物和人类与CNTs等碳纳米材料接触的机会也越来越大,对CNTs的生物安全性进行研究和评价就显得十分必要,而且已有研究表明CNTs确实会对生物体产生毒性作用。虽然有关CNTs的生物安全性研究已经取得了一定的进展,但对于其产生毒性作用的机理还需要进行更加深入的探究。众所周知,游离的钙离子作为第二信使在细胞信号转导过程中起着非常重要的作用,是维持生物体正常生命活动的必须营养元素。研究CNTs对细胞钙离子代谢的影响,对于揭示CNTs产生生物毒性的机理,准确评价CNTs生物安全性具有重要的意义。本文主要采用钙离子选择性微电极技术研究了在多壁碳纳米管(Multi-walled Carbon Nanotubes,MWCNTs)作用下芦荟细胞原生质体的钙离子释放问题。钙离子选择性微电极技术可以实现无损伤的原位实时测量,也无需像荧光探针方法那样对细胞进行紫外照射,因此可以长时间连续测量。论文首先对钙离子选择性微电极的制备过程及其性能进行了研究,对于电极拉制温度、硅烷化温度和硅烷化试剂用量等制备条件进行了优化,成功制备了性能优良的钙离子选择性微电极。经测试,电极的转化效率在大于90%,当钙离子浓度在10-5~10-1 mol/L范围内时,电极的响应时间小于1 s。在此基础上,对MWCNTs作用下芦荟细胞原生质体的钙离子释放进行了研究。实验中所用的芦荟细胞原生质体通过酶解法制取,用FDA法检测其活性。MWCNTs在使用前进行纯化处理,XRD测试未发现催化剂残留。经酶解法提取的原生质体先用无钙培养基进行洗涤,使背景钙离子浓度降至约10-5 mol/L,之后在含有不同浓度和不同直径MWCNTs的无钙培养基中进行培养,并连续测量原生质体释放到培养基中的钙离子浓度。结果发现,与相同条件下培养的对照组对比,MWCNTs的加入加速了原生质体钙离子的释放,使得培养基中的钙离子浓度在较短的时间内达到饱和。当MWCNTs浓度在20 mg/L~40 mg/L范围时,随着MWCNTs浓度的增加,钙离子释放速度加快;而当MWCNTs的浓度达到50 mg/L时,钙离子释放速度略有降低。对两种不同直径的MWCNTs进行比较,直径为20-30 nm的MWCNTs较直径小于8 nm的MWCNTs对钙离子释放的加速作用更为显著。上述结果从一个新的层面揭示了MWCNTs对植物细胞的影响,对全面理解MWCNTs与植物细胞间的相互作用形式和作用机理,以及对MWCNTs的生物安全性进行评价具有重要作用。
[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.
【学位授予单位】：东北师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：TB383.1

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