本文選題：富勒醇　切入點(diǎn)：細(xì)胞核靶向　出處：《蘇州大學(xué)》2012年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景：電離輻射作用于機(jī)體，產(chǎn)生大量的自由基如H~.、H_2、H_2O_2、 H_(aq)-、OH.、e_(aq)-等，這些自由基對(duì)機(jī)體造成嚴(yán)重的損傷。Templeteton等的研究證實(shí)低LET射線造成的電離損傷中，90%是由OH.引起的。OH.使DNA雙鏈斷裂，導(dǎo)致染色體畸變以及遺傳物質(zhì)丟失，所以DNA雙鏈斷裂（DSB）是DNA分子結(jié)構(gòu)中最關(guān)鍵的損傷，也是細(xì)胞死亡的主要原因。因此理想的輻射防護(hù)藥物應(yīng)該能夠進(jìn)入細(xì)胞內(nèi)，特別是細(xì)胞核內(nèi)，在電離輻射產(chǎn)生自由基的瞬間立即清除DNA周?chē)淖杂苫�。盡管人體中有一些天然存在的自由基清除劑（如超氧化物歧化酶SOD、谷胱甘肽過(guò)氧化物酶、谷胱甘肽還原酶等），，但是這類自由基清除劑無(wú)法清除H_(aq)-、OH.等自由基，并且不能穿透生物膜，進(jìn)入到細(xì)胞核內(nèi)。 富勒烯的毒理學(xué)研究證明它是低毒化合物，其水溶性衍生物富勒醇具有良好的自由基清除作用，尤其是與OH~.、e_(aq)-的反應(yīng)速率常數(shù)可達(dá)（0.5~3.3）×1010M~(-1)S~(-1)。水溶性富勒醇能夠快速穿過(guò)細(xì)胞膜，到達(dá)細(xì)胞內(nèi)，并且主要分布在胞漿以及線粒體等細(xì)胞器中。目前，國(guó)內(nèi)外相關(guān)研究主要是將富勒烯聯(lián)接上不同的化學(xué)基團(tuán)，形成新的富勒烯衍生物，從而得到廣泛應(yīng)用。但是，迄今為止，仍然沒(méi)有富勒烯及其衍生物能夠被載帶并進(jìn)入到細(xì)胞核內(nèi)，作為輻射防護(hù)劑或者自由基清除劑的報(bào)道。 目的：建立一種新型的細(xì)胞核靶向納米載藥系統(tǒng)，能夠高效載帶富勒醇到達(dá)細(xì)胞質(zhì)以及細(xì)胞核內(nèi)，清除電離輻射產(chǎn)生的自由基，從而為研發(fā)輻射防護(hù)藥物提供實(shí)驗(yàn)數(shù)據(jù)。 方法：以四丁基氫氧化銨（TBAH）催化法合成富勒醇，通過(guò)傅立葉變換紅外光譜、核磁共振氫譜以及質(zhì)譜檢測(cè)對(duì)其進(jìn)行鑒定和表征。采用高壓乳勻法制備新型細(xì)胞核靶向富勒醇固體脂質(zhì)納米粒C60(OH)_(24)-NLC-E，其表面連接與核受體有高度親和力的己烯雌酚，通過(guò)納米粒度儀檢測(cè)其大小及分布，掃描電鏡觀察其微觀形態(tài)。使用激光共聚焦顯微鏡動(dòng)態(tài)觀察包裹羅丹明-6G的C60(OH)_(24)-NLC-E進(jìn)入細(xì)胞的過(guò)程。通過(guò)MTT比色法測(cè)定C60(OH)_(24)-NLC-E對(duì)V79細(xì)胞的毒性作用；通過(guò)細(xì)胞克隆形成實(shí)驗(yàn)觀察該納米粒對(duì)V79細(xì)胞的輻射防護(hù)作用；通過(guò)對(duì)V79細(xì)胞進(jìn)行γ-H2AX焦點(diǎn)分析，觀察其對(duì)DNA分子的輻射防護(hù)作用。 結(jié)果：（1）、以胺催化合成法制備富勒醇，對(duì)新合成的富勒醇進(jìn)行紅外光譜分析，在3433cm~(-1)處可見(jiàn)強(qiáng)而寬的羥基吸收峰，又進(jìn)行氫核磁共振譜分析，可見(jiàn)δ=3.34處為氘代DMSO中含有雜質(zhì)水所產(chǎn)生的水峰，δ=2.50處為DMSO溶劑吸收峰，δ=1.24處為羥基特征峰，進(jìn)一步進(jìn)行質(zhì)譜分析，m/z=720的主峰表示C60分子量，此外，在720-1128范圍內(nèi)的峰，平均間隔為68，恰好為4個(gè)羥基的分子量，在m/z=1128處的峰為C60(OH)x的準(zhǔn)分子離子峰，與C60(OH)_(24)的分子量相符。（2）、對(duì)于新制備的納米脂質(zhì)載體C60(OH)_(24)-NLC-E，經(jīng)過(guò)檢測(cè)可見(jiàn)其形態(tài)近似球形，大小較為均一，直徑大約300nm左右。通過(guò)激光共聚焦顯微鏡觀察包裹羅丹明-6G的C60(OH)_(24)-NLC-E，能夠快速進(jìn)入細(xì)胞，甚至細(xì)胞核內(nèi)，15~30min達(dá)到飽和。（3）、當(dāng)C60(OH)24-NLC-E濃度1.5μmol/L時(shí)，細(xì)胞存活率超過(guò)95%。（4）、經(jīng)過(guò)60Co-γ射線照射0~8Gy時(shí)，于照射前30min加入C60(OH)_(24)-NLC-E（1.27μmol/L）的加藥組細(xì)胞生存率明顯大于單純照射組（p0.05），并且加藥組的D_0和D_q值也明顯高于單純照射組（p0.05）。（5）、于照射前30min加入C60(OH)_(24)-NLC-E的加藥組在照射后30min的γ-H2AX焦點(diǎn)數(shù)明顯低于單純照射的對(duì)照組細(xì)胞（p0.05）。 結(jié)論：成功研制了C60(OH)_(24)-NLC-E，能夠靶向且高效地載帶富勒醇到達(dá)細(xì)胞漿和細(xì)胞核，有效地清除自由基，直接保護(hù)DNA免受輻射損傷，具有低毒性和高效輻射防護(hù)的特點(diǎn)。
[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.
緇撴灉錛

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