细胞核靶向富勒醇固体脂质纳米粒的辐射防护作用

发布时间：2018-03-19 15:08

  本文选题：富勒醇　切入点：细胞核靶向　出处：《苏州大学》2012年硕士论文　论文类型：学位论文

【摘要】：研究背景：电离辐射作用于机体，产生大量的自由基如H~.、H_2、H_2O_2、 H_(aq)-、OH.、e_(aq)-等，这些自由基对机体造成严重的损伤。Templeteton等的研究证实低LET射线造成的电离损伤中，90%是由OH.引起的。OH.使DNA双链断裂，导致染色体畸变以及遗传物质丢失，所以DNA双链断裂（DSB）是DNA分子结构中最关键的损伤，也是细胞死亡的主要原因。因此理想的辐射防护药物应该能够进入细胞内，特别是细胞核内，在电离辐射产生自由基的瞬间立即清除DNA周围的自由基。尽管人体中有一些天然存在的自由基清除剂（如超氧化物歧化酶SOD、谷胱甘肽过氧化物酶、谷胱甘肽还原酶等），，但是这类自由基清除剂无法清除H_(aq)-、OH.等自由基，并且不能穿透生物膜，进入到细胞核内。 富勒烯的毒理学研究证明它是低毒化合物，其水溶性衍生物富勒醇具有良好的自由基清除作用，尤其是与OH~.、e_(aq)-的反应速率常数可达（0.5~3.3）×1010M~(-1)S~(-1)。水溶性富勒醇能够快速穿过细胞膜，到达细胞内，并且主要分布在胞浆以及线粒体等细胞器中。目前，国内外相关研究主要是将富勒烯联接上不同的化学基团，形成新的富勒烯衍生物，从而得到广泛应用。但是，迄今为止，仍然没有富勒烯及其衍生物能够被载带并进入到细胞核内，作为辐射防护剂或者自由基清除剂的报道。 目的：建立一种新型的细胞核靶向纳米载药系统，能够高效载带富勒醇到达细胞质以及细胞核内，清除电离辐射产生的自由基，从而为研发辐射防护药物提供实验数据。 方法：以四丁基氢氧化铵（TBAH）催化法合成富勒醇，通过傅立叶变换红外光谱、核磁共振氢谱以及质谱检测对其进行鉴定和表征。采用高压乳匀法制备新型细胞核靶向富勒醇固体脂质纳米粒C60(OH)_(24)-NLC-E，其表面连接与核受体有高度亲和力的己烯雌酚，通过纳米粒度仪检测其大小及分布，扫描电镜观察其微观形态。使用激光共聚焦显微镜动态观察包裹罗丹明-6G的C60(OH)_(24)-NLC-E进入细胞的过程。通过MTT比色法测定C60(OH)_(24)-NLC-E对V79细胞的毒性作用；通过细胞克隆形成实验观察该纳米粒对V79细胞的辐射防护作用；通过对V79细胞进行γ-H2AX焦点分析，观察其对DNA分子的辐射防护作用。 结果：（1）、以胺催化合成法制备富勒醇，对新合成的富勒醇进行红外光谱分析，在3433cm~(-1)处可见强而宽的羟基吸收峰，又进行氢核磁共振谱分析，可见δ=3.34处为氘代DMSO中含有杂质水所产生的水峰，δ=2.50处为DMSO溶剂吸收峰，δ=1.24处为羟基特征峰，进一步进行质谱分析，m/z=720的主峰表示C60分子量，此外，在720-1128范围内的峰，平均间隔为68，恰好为4个羟基的分子量，在m/z=1128处的峰为C60(OH)x的准分子离子峰，与C60(OH)_(24)的分子量相符。（2）、对于新制备的纳米脂质载体C60(OH)_(24)-NLC-E，经过检测可见其形态近似球形，大小较为均一，直径大约300nm左右。通过激光共聚焦显微镜观察包裹罗丹明-6G的C60(OH)_(24)-NLC-E，能够快速进入细胞，甚至细胞核内，15~30min达到饱和。（3）、当C60(OH)24-NLC-E浓度1.5μmol/L时，细胞存活率超过95%。（4）、经过60Co-γ射线照射0~8Gy时，于照射前30min加入C60(OH)_(24)-NLC-E（1.27μmol/L）的加药组细胞生存率明显大于单纯照射组（p0.05），并且加药组的D_0和D_q值也明显高于单纯照射组（p0.05）。（5）、于照射前30min加入C60(OH)_(24)-NLC-E的加药组在照射后30min的γ-H2AX焦点数明显低于单纯照射的对照组细胞（p0.05）。 结论：成功研制了C60(OH)_(24)-NLC-E，能够靶向且高效地载带富勒醇到达细胞浆和细胞核，有效地清除自由基，直接保护DNA免受辐射损伤，具有低毒性和高效辐射防护的特点。
[Abstract]:Background: the effect of ionizing radiation on the body, free such as H~., H_2, H_2O_2 radicals, H_ (AQ) - OH., e_ - (AQ), the research of these free radicals caused serious damage to the body of.Templeteton confirmed by LET radiation and low ionization damage, which is caused by OH. 90% the.OH. to DNA double strand breaks lead to chromosome aberration and genetic material loss, so DNA double strand breaks (DSB) is the most critical damage in the molecular structure of DNA, and is a major cause of cell death. Therefore, radiation protection ideal drug should be able to enter the cell, especially in the nucleus, produce free radicals in ionizing radiation at the moment of clearance around DNA radicals. Though the body has some natural free radical scavenger (such as superoxide dismutase SOD and glutathione peroxidase, glutathione reductase), but this kind of free radical scavenger can not be removed H_ (AQ) -, OH., and other free radicals, and can not penetrate the biofilm into the nucleus.
It is proved that the toxicological study of fullerene compound toxicity, its water soluble derivatives of Fuller alcohol has good effects on scavenging free radical, especially with OH~., e_ (AQ) - the reaction rate constant is (0.5~3.3) * 1010M~ (-1) S~ (-1). Water soluble Fuller alcohol can quickly through the cell membrane. Arrive in the cell, and is mainly distributed in the cytoplasm and mitochondria. At present, the domestic and foreign related research is mainly connected with different chemical groups of fullerenes, the formation of new fullerene derivatives, which are widely used. However, so far, still no fullerene and its derivatives can be loaded into the nucleus inside, as a radiation protective agent or free radical scavenger is reported.
Objective: to establish a model of the target nucleus to the nano drug carrier system, can be efficient with alcohol to Fuller cytoplasm and nucleus, removal of ionizing radiation generated free radicals, so as to provide experimental data for the development of radiation protection drug.
Methods: four Butyl Ammonium Hydroxide (TBAH) catalytic synthesis of alcohol by Fu Liye Fuller, FT-IR, 1H NMR and mass spectrometry for identification and characterization. Prepared by high-pressure homogenization of new nuclear target nanoparticles to Fuller solid lipid C60 alcohol (OH) _ (24) -NLC-E, the surface the connection of diethylstilbestrol with high affinity to the nuclear receptor, the size and distribution of detection by nanoparticle size analyzer, scanning electron microscopy to observe the micro morphology. Using laser confocal microscopy observation of dynamic package Luo Danming -6G C60 (OH) _ (24) -NLC-E into the cell process. Through the determination of C60 MTT colorimetric method (OH) _ (24) toxic effects of -NLC-E on V79 cells; the cell colony formation experiment to observe the protective effect of the nanoparticles radiation on V79 cells; analyzed by gamma -H2AX focus on V79 cells, observe the radioprotective effects of DNA molecule.
缁撴灉锛

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