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基于膽固醇的陽離子脂質(zhì)材料的設(shè)計(jì)合成及其在基因轉(zhuǎn)染中的應(yīng)用研究

發(fā)布時(shí)間:2018-09-18 18:00
【摘要】:目的:基因治療是目前癌癥和遺傳性相關(guān)疾病治療中很有前途的一種新技術(shù),其關(guān)鍵問題在于構(gòu)建有效攜載治療基因并使其發(fā)揮作用的載體。非病毒載體作為新興的基因轉(zhuǎn)導(dǎo)系統(tǒng),具有低毒、低免疫原性、無基因插入片段大小的限制,以及制備方便、便于保存和使用等優(yōu)勢。陽離子脂質(zhì)體是非病毒載體中最常用的載體,具有易于生產(chǎn)、低免疫原性、無致癌性和保護(hù)基因免受核酸酶降解等優(yōu)點(diǎn),在介導(dǎo)基因轉(zhuǎn)染上具有廣闊的發(fā)展前景。本論文綜合考慮膽固醇疏水基團(tuán)、不同種類胺基和堿性氨基酸頭部的片段優(yōu)勢,通過連接鍵的橋連,在其側(cè)鏈引入不同的陽離子頭部,設(shè)計(jì)合成了三個(gè)系列含有不同胺基或堿性氨基酸殘基的膽固醇陽離子脂質(zhì)材料,為陽離子脂質(zhì)體的制備及基因治療提供載體材料。方法:以膽固醇,1,6-己二醇,堿性氨基酸(賴氨酸、組氨酸和精氨酸),二甲胺鹽酸鹽,二乙胺,三乙胺,1,4-二溴乙醚,1,4-二溴丁烷,1,6-二溴己烷,1,12-二溴十二烷等為原料,設(shè)計(jì)合成了單胺基頭部(M1-M6)、堿性氨基酸頭部(A1-A6)和雙生脂質(zhì)(G1-G4)三個(gè)系列的陽離子脂質(zhì)材料。通過質(zhì)譜、核磁、紅外等方法對合成的材料進(jìn)行結(jié)構(gòu)鑒定。采用薄膜分散法將上述陽離子脂質(zhì)材料與輔助脂質(zhì)DOPE混合制備得到陽離子脂質(zhì)體,并進(jìn)行表征。采用MTT比色法對不同空白陽離子脂質(zhì)體的細(xì)胞毒性進(jìn)行檢測。采用凝膠電泳阻滯實(shí)驗(yàn)考察不同陽離子脂質(zhì)體與p EGFP質(zhì)粒的結(jié)合能力,篩選出DNA與陽離子脂質(zhì)體完全復(fù)合時(shí)的N/P比,并進(jìn)行體外轉(zhuǎn)染活性的篩選。采用流式細(xì)胞儀定量檢測細(xì)胞轉(zhuǎn)染效率,并用熒光顯微鏡定性觀察p EGFP在細(xì)胞內(nèi)的表達(dá)情況。對初篩轉(zhuǎn)染活性較好的陽離子脂質(zhì)材料進(jìn)行處方的優(yōu)化,篩選出的最優(yōu)處方進(jìn)一步考察血清存在對其細(xì)胞轉(zhuǎn)染活性的影響。結(jié)果:設(shè)計(jì)合成了三系列共16種基于膽固醇的陽離子脂質(zhì)材料,經(jīng)1H NMR,MS,IR等方法鑒定,所得化合物的結(jié)構(gòu)與目標(biāo)物的結(jié)構(gòu)一致。經(jīng)納米粒度及Zeta電位分析儀測定,所制備的16種陽離子脂質(zhì)體粒徑均小于240 nm,Zeta電位均在35 m V以上,滿足基因載體的要求。MTT細(xì)胞毒性實(shí)驗(yàn)結(jié)果表明,上述陽離子脂質(zhì)體IC50均大于陽性對照Lipofectamine 2000(IC50=33μM);與DC-Chol(IC50=51μM)相比,除G1(IC50=44μM)、G4(IC50=35μM)、A4(IC50=48μM)和A5(IC50=49μM)外,其它12種陽離子脂質(zhì)材料所得脂質(zhì)體IC50均大于DC-Chol,安全性和生物相容性好。凝膠阻滯實(shí)驗(yàn)結(jié)果顯示,在低N/P比(小于3)的情況下,所合成的陽離子脂質(zhì)材料都具有較強(qiáng)的攜載DNA的能力;蜣D(zhuǎn)染實(shí)驗(yàn)結(jié)果表明,除M3、M5和G1外,其余13種陽離子脂質(zhì)材料所得脂質(zhì)體均具有良好的轉(zhuǎn)染活性。M1、M6和A1-A6的轉(zhuǎn)染活性優(yōu)于DC-Chol(p0.05或0.01),其中M6、A4和A6的轉(zhuǎn)染活性最好,M6和A4轉(zhuǎn)染效率與Lipofectamine 2000相當(dāng)(p0.05),A6明顯優(yōu)于Lipofectamine2000(p0.05)。脂質(zhì)材料M1,M6和A1-A3的處方優(yōu)化和血清存在下的轉(zhuǎn)染實(shí)驗(yàn)結(jié)果表明,即使在血清存在下M1和M6仍具有很好的轉(zhuǎn)染活性,優(yōu)于Lipofectamine2000(p0.05或0.01)。結(jié)論:本論文在膽固醇骨架中引入不同胺基頭部構(gòu)建了三個(gè)系列16種陽離子脂質(zhì)材料并對其細(xì)胞毒性和基因轉(zhuǎn)染活性進(jìn)行研究。研究結(jié)果顯示,上述陽離子脂質(zhì)材料所得基因載體具有粒徑均一,細(xì)胞毒性小,攜載DNA能力強(qiáng)等優(yōu)點(diǎn)。除M3、M5和G1外,所有陽離子脂質(zhì)體均具有較好的轉(zhuǎn)染活性,其中M1和M6的活性最好,在血清存在下仍優(yōu)于DC-Chol和Lipofectamine 2000。通過進(jìn)一步的體內(nèi)活性研究,可望為基因治療提供安全有效的載體。
[Abstract]:Objective: Gene therapy is a promising new technique in the treatment of cancer and hereditary related diseases. The key problem is to construct effective carriers for carrying therapeutic genes and making them work. Cationic liposomes are the most commonly used carriers in non-viral vectors. They have the advantages of easy production, low immunogenicity, non-carcinogenicity and protection of genes from nuclease degradation. They have broad prospects for gene transfection. Three series of cholesterol cationic lipids containing different amino or basic amino acid residues were designed and synthesized by bridging the amino and basic amino acid heads and introducing different cationic heads into their side chains. Three series of amino acids (lysine, histidine and arginine), dimethylamine hydrochloride, diethylamine, triethylamine, 1,4-dibromoethyl ether, 1,4-dibromobutane, 1,6-dibromohexane, 1,12-dibromododecane) were designed and synthesized from cholesterol, 1,6-hexanediol, basic amino acid head (A1-A6) and Gemini lipid (G1-G4). Cationic Liposomes. Structural identification of the synthesized materials was carried out by mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. Cationic liposomes were prepared by mixing the cationic liposomes with auxiliary liposomes DOPE by membrane dispersion method and characterized. The cytotoxicity of different blank cationic liposomes was detected by MTT colorimetry. The binding ability of different cationic liposomes to P EGFP plasmid was examined by gel electrophoresis block test. The N/P ratio of DNA completely combined with cationic liposomes was screened, and the transfection activity was screened. The transfection efficiency was quantitatively detected by flow cytometry, and the expression of P EGFP in cells was qualitatively observed by fluorescence microscope. Results: Three series of 16 cholesterol-based cationic lipid materials were designed and synthesized and identified by 1H NMR, MS and IR. The sixteen cationic liposomes were found to be smaller than 240 nm in size and above 35 m V in Zeta potential. The results of MTT cytotoxicity test showed that the IC50 of these liposomes was higher than that of Lipofectamine 2000 (IC50 = 33 mu M). Compared with DC-Chol (IC50=51 M), except G1 (IC50=44 M M), G4 (IC50=35 M), A4 (IC50=48 mul), and (12), the liposomes of other 3 cationic liposomes were all larger than that of the liposomes, and their safety and biocompatibility were good. The results of gel retardation test showed that the synthesized cationic lipids were low to low ratio (less than 3). The transfection activity of M1, M6 and A 1-A6 was better than that of DC-Chol (p0.05 or 0.01). The transfection activity of M6, A4 and A6 was the best, and the transfection efficiency of M6 and A4 was the same as that of Lipofectamine 2000. When (p0.05), A6 was significantly superior to Lipofectamine 2000 (p0.05). The results of formulation optimization and serum transfection of lipid materials M1, M6 and A1-A3 showed that M1 and M6 had good transfection activity even in the presence of serum, superior to Lipofectamine 2000 (p0.05 or 0.01). Conclusion: Different amino groups were introduced into the cholesterol skeleton in this study. Three series of 16 cationic liposomes were constructed and their cytotoxicity and gene transfection activity were studied. The results showed that the cationic liposomes had the advantages of uniform particle size, low cytotoxicity and strong ability to carry DNA. Except M3, M5 and G1, all cationic liposomes had good transfection activity. Among them, M1 and M6 have the best activity and are still superior to DC-Chol and Lipofectamine 2000 in the presence of serum.
【學(xué)位授予單位】:第四軍醫(yī)大學(xué)
【學(xué)位級別】:碩士
【學(xué)位授予年份】:2015
【分類號】:R943

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王冰;張樹彪;周集體;趙不凋;楊寶靈;崔紹輝;趙軼男;;陽離子脂質(zhì)體介導(dǎo)的基因轉(zhuǎn)移機(jī)制[J];中國組織工程研究與臨床康復(fù);2011年08期

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本文編號:2248683

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