自组装Trolox-壳聚糖纳米颗粒在氧化应激条件下对秀丽隐杆线虫的保护作用

发布时间：2018-01-22 04:59

本文关键词： Trolox-壳聚糖纳米颗粒秀丽隐杆线虫氧化应激　出处：《贵州师范大学》2014年硕士论文　论文类型：学位论文

【摘要】：壳聚糖纳米颗粒具有良好的分子负载功能，因而被广泛用作基因、蛋白质以及小分子药物等生物活性物质的输运载体。由于纳米壳聚糖制备等技术原因的局限，目前药用的纳米壳聚糖载体粒径大多在100nm以上，绝少有小于50nm的应用实例。然而，考虑到纳米颗粒在生物体内的吸收与利用与其尺寸有着密切的关联，粒径越小往往越能促进生物活性分子的有效吸收。鉴于此，本论文从小粒径Trolox-壳聚糖纳米颗粒的制备入手，，探索了它对秀丽隐杆线虫在应激条件下抗衰老效果及研究机制。研究所得的具体实验结果如下： 1、小粒径Trolox-壳聚糖纳米颗粒的制备、表征与生物相容性研究通过氧化降解方法可直接获得小分子量壳聚糖，随后加入环氧乙烷进行羟乙基化修饰，自组装形成水溶性的纳米颗粒。经透射电镜和原子力显微镜对该纳米颗粒进行形态表征，结果显示粒径分布均匀，约为30nm；根据实验室前期的研究结果，所制备的壳聚糖自组装纳米颗粒具有无毒、生物相容性好的特点。此外，尽管抗氧化剂Trolox自称水溶性维生素E，但其水中溶解度仍然十分有限，这直接影响到它的生物利用度。本研究将Trolox自组装在水溶性壳聚糖颗粒之内部，所获得的Trolox-壳聚糖纳米颗粒在水溶液中的溶解度较Trolox单体提高27倍之多，也体现出该纳米颗粒对小分子物质的高负载效率。 2、百草枯Paraquat氧化损伤秀丽隐杆线虫模型的建立为验证Trolox-壳聚糖纳米颗粒的抗氧化活性，首先建立了1mmol/L百草枯Paraquat诱导秀丽隐杆线虫氧化应激的实验模型。当秀丽隐杆线虫处于氧化应激状态时，体内自由基含量便会高于正常生理水平，抗氧化平衡则被打破，促使氧化应激的产生，引起生物大分子(例如蛋白、脂质和DNA)的损伤，进而引起细胞死亡，最终导致一系列氧化应激相关的病理过程。 3、Trolox-壳聚糖自组装纳米颗粒对氧化应激条件下秀丽隐杆线虫的保护作用 (1)对线虫寿命与后代数目影响的实验结果显示：在氧化应激条件下，Trolox-壳聚糖纳米颗粒比Trolox单体对线虫具有更佳的保护作用，能进一步延长线虫寿命；无论是正常生长还是氧化应激条件下，Trolox-壳聚糖纳米颗粒均可以增加线虫后代数目； (2)对线虫运动能力与进食能力影响的实验结果显示：在氧化应激条件下，Trolox-壳聚糖纳米颗粒对线虫运动能力与进食能力的保护作用在第四天时分别达到最大值，比空白对照组CTRL组分别增加了42.64%与345%，比Trolox组分别增加了22.67%与50.85%； (3)对线虫体内活性氧自由基ROS与脂褐素清除能力影响的实验结果显示：在氧化应激条件下，Trolox-壳聚糖纳米颗粒比Trolox单体能更好地清除线虫体内的活性氧自由基ROS与脂褐素，更好地保护线虫。 (4)对线虫体内DAF-16蛋白定位影响的实验结果显示：在氧化应激条件下，通过观察突变型线虫TJ356体内DAF-16蛋白在细胞质、质核间、细胞核中的分布，结果表明，Trolox-壳聚糖纳米颗粒比Trolox单体能进一步地促进DAF-16蛋白向细胞核转移，提高在细胞核内的分布率。
[Abstract]:Chitosan nanoparticles have good molecular load function, so it has been widely used as gene, protein and small molecule drugs and other bioactive substances transport carrier. The nano chitosan preparation technology causes the limitations of current medical nano chitosan carrier particle size more than 100nm, there are few examples of less than 50nm. However, considering the nanoparticles in vivo absorption and utilization of its dimensions are closely related, the smaller the particle size is often more effective to promote the absorption of biologically active molecules. In view of this, this thesis starts from the preparation of Trolox- particle size of chitosan nanoparticles and explored it on anti-aging effect and Study on the mechanism of Caenorhabditis elegans under stress. The experimental results are as follows:
1, preparation, characterization and biocompatibility of small particle size Trolox- chitosan nanoparticles
The method can be directly obtained by oxidative degradation of low molecular weight chitosan, followed by addition of ethylene oxide hydroxyethylation modification, self-assembly of water-soluble nanoparticles. By transmission electron microscopy and atomic force microscopy were used to characterize the morphology of nanoparticles showed uniform particle size distribution, about 30nm; based on the results of previous and the prepared chitosan self-assembled nanoparticles with non-toxic, biocompatible. In addition, despite claiming antioxidant Trolox water soluble vitamin E, but its water solubility is still very limited, which directly affect its bioavailability. In this study, Trolox self-assembly in water soluble inner shell chitosan particles, Trolox- chitosan nanoparticles obtained solubility in aqueous solution with Trolox monomer increased 27 times, but also reflects the high load effect of the nanoparticles on small molecules Rate.
2, the establishment of a Paraquat oxidative damage model of Caenorhabditis elegans
In order to verify the antioxidant activity of Trolox- chitosan nanoparticles, an experimental model of oxidative stress induced by 1mmol/L paraquat Paraquat in the induction of Caenorhabditis elegans was established.
When the Caenorhabditis elegans undergoing oxidative stress, free radicals will be higher than the normal physiological level, antioxidant balance is broken, the oxidative stress, caused by biological macromolecules (such as protein, lipid and DNA) damage, causing cell death, resulting in a series of oxidative stress related pathological process.
3, the protective effect of Trolox- chitosan self assembled nanoparticles on Caenorhabditis elegans under oxidative stress
(1) influence on the life of c.elegance and offspring number of experimental results show that: in the condition of oxidative stress, Trolox- chitosan nanoparticles than Trolox monomer has better protective effect on nematodes, can further extend the lifespan of C.elegans; either normal growth or oxidative stress conditions, Trolox- chitosan nanoparticles can increase nematode progeny the number of;
(2) influence on the movement ability and the ability of the nematode feeding experiments showed that under oxidative stress conditions, the protective effect of Trolox- chitosan nanoparticles on the nematode movement ability and feeding capability respectively reached the maximum in fourth days, compared with the blank control group and CTRL group were increased by 42.64% and 345%, than in the Trolox group were increased 22.67% and 50.85%;
(3) of nematodes. Active oxygen free radical scavenging ability of ROS and lipofuscin influence the experimental results showed that: in the condition of oxidative stress, reactive oxygen free radicals of ROS and lipofuscin Trolox- chitosan nanoparticles than Trolox monomer can better remove the worms, to better protect the worm.
(4) the localization of DAF-16 protein of nematodes. The experimental results showed that: in the condition of oxidative stress, by observing the mutant nematode TJ356 in vivo of DAF-16 protein in the cytoplasm, nucleus, nucleus of the distribution, the results show that the Trolox- chitosan nanoparticles than Trolox monomer can further promote the DAF-16 protein translocates to the nucleus, improve the distribution in the nucleus.

【学位授予单位】：贵州师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：TQ460.1;R965

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