本文關(guān)鍵詞： Trolox-殼聚糖納米顆粒 秀麗隱桿線蟲 氧化應(yīng)激　出處：《貴州師范大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：殼聚糖納米顆粒具有良好的分子負(fù)載功能，因而被廣泛用作基因、蛋白質(zhì)以及小分子藥物等生物活性物質(zhì)的輸運(yùn)載體。由于納米殼聚糖制備等技術(shù)原因的局限，目前藥用的納米殼聚糖載體粒徑大多在100nm以上，絕少有小于50nm的應(yīng)用實(shí)例。然而，考慮到納米顆粒在生物體內(nèi)的吸收與利用與其尺寸有著密切的關(guān)聯(lián)，粒徑越小往往越能促進(jìn)生物活性分子的有效吸收。鑒于此，本論文從小粒徑Trolox-殼聚糖納米顆粒的制備入手，，探索了它對(duì)秀麗隱桿線蟲在應(yīng)激條件下抗衰老效果及研究機(jī)制。研究所得的具體實(shí)驗(yàn)結(jié)果如下： 1、小粒徑Trolox-殼聚糖納米顆粒的制備、表征與生物相容性研究 通過氧化降解方法可直接獲得小分子量殼聚糖，隨后加入環(huán)氧乙烷進(jìn)行羥乙基化修飾，自組裝形成水溶性的納米顆粒。經(jīng)透射電鏡和原子力顯微鏡對(duì)該納米顆粒進(jìn)行形態(tài)表征，結(jié)果顯示粒徑分布均勻，約為30nm；根據(jù)實(shí)驗(yàn)室前期的研究結(jié)果，所制備的殼聚糖自組裝納米顆粒具有無毒、生物相容性好的特點(diǎn)。此外，盡管抗氧化劑Trolox自稱水溶性維生素E，但其水中溶解度仍然十分有限，這直接影響到它的生物利用度。本研究將Trolox自組裝在水溶性殼聚糖顆粒之內(nèi)部，所獲得的Trolox-殼聚糖納米顆粒在水溶液中的溶解度較Trolox單體提高27倍之多，也體現(xiàn)出該納米顆粒對(duì)小分子物質(zhì)的高負(fù)載效率。 2、百草枯Paraquat氧化損傷秀麗隱桿線蟲模型的建立 為驗(yàn)證Trolox-殼聚糖納米顆粒的抗氧化活性，首先建立了1mmol/L百草枯Paraquat誘導(dǎo)秀麗隱桿線蟲氧化應(yīng)激的實(shí)驗(yàn)?zāi)Ｐ汀?當(dāng)秀麗隱桿線蟲處于氧化應(yīng)激狀態(tài)時(shí)，體內(nèi)自由基含量便會(huì)高于正常生理水平，抗氧化平衡則被打破，促使氧化應(yīng)激的產(chǎn)生，引起生物大分子(例如蛋白、脂質(zhì)和DNA)的損傷，進(jìn)而引起細(xì)胞死亡，最終導(dǎo)致一系列氧化應(yīng)激相關(guān)的病理過程。 3、Trolox-殼聚糖自組裝納米顆粒對(duì)氧化應(yīng)激條件下秀麗隱桿線蟲的保護(hù)作用 (1)對(duì)線蟲壽命與后代數(shù)目影響的實(shí)驗(yàn)結(jié)果顯示：在氧化應(yīng)激條件下，Trolox-殼聚糖納米顆粒比Trolox單體對(duì)線蟲具有更佳的保護(hù)作用，能進(jìn)一步延長(zhǎng)線蟲壽命；無論是正常生長(zhǎng)還是氧化應(yīng)激條件下，Trolox-殼聚糖納米顆粒均可以增加線蟲后代數(shù)目； (2)對(duì)線蟲運(yùn)動(dòng)能力與進(jìn)食能力影響的實(shí)驗(yàn)結(jié)果顯示：在氧化應(yīng)激條件下，Trolox-殼聚糖納米顆粒對(duì)線蟲運(yùn)動(dòng)能力與進(jìn)食能力的保護(hù)作用在第四天時(shí)分別達(dá)到最大值，比空白對(duì)照組CTRL組分別增加了42.64%與345%，比Trolox組分別增加了22.67%與50.85%； (3)對(duì)線蟲體內(nèi)活性氧自由基ROS與脂褐素清除能力影響的實(shí)驗(yàn)結(jié)果顯示：在氧化應(yīng)激條件下，Trolox-殼聚糖納米顆粒比Trolox單體能更好地清除線蟲體內(nèi)的活性氧自由基ROS與脂褐素，更好地保護(hù)線蟲。 (4)對(duì)線蟲體內(nèi)DAF-16蛋白定位影響的實(shí)驗(yàn)結(jié)果顯示：在氧化應(yīng)激條件下，通過觀察突變型線蟲TJ356體內(nèi)DAF-16蛋白在細(xì)胞質(zhì)、質(zhì)核間、細(xì)胞核中的分布，結(jié)果表明，Trolox-殼聚糖納米顆粒比Trolox單體能進(jìn)一步地促進(jìn)DAF-16蛋白向細(xì)胞核轉(zhuǎn)移，提高在細(xì)胞核內(nèi)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.

【學(xué)位授予單位】：貴州師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：TQ460.1;R965

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 劉恩岐,倉(cāng)林讓;模型動(dòng)物——秀麗隱桿線蟲研究進(jìn)展[J];動(dòng)物科學(xué)與動(dòng)物醫(yī)學(xué);2003年10期

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