陽離子多肽—硬脂酸嫁接物基因給藥系統(tǒng)研究
發(fā)布時(shí)間：2018-11-05 11:21
【摘要】：陽離子多肽-硬脂酸嫁接物具有較豐富的正電荷,可通過靜電作用與質(zhì)粒DNA (plasmid DNA, pDNA)進(jìn)行復(fù)合,為實(shí)現(xiàn)有效的體外基因轉(zhuǎn)染和體內(nèi)抗腫瘤療效提供了極大的可能性。本研究構(gòu)建了陽離子多肽-硬脂酸/pDNA復(fù)合物和聚乙二醇修飾多肽-硬脂酸/pDNA復(fù)合物基因給藥系統(tǒng),考察了其基本理化性質(zhì),體外轉(zhuǎn)染效率,體內(nèi)抗腫瘤療效以及細(xì)胞內(nèi)涵體逃逸過程。 陽離子多肽-硬脂酸嫁接物(cationic peptide stearate, STR-Pep)系通過多肽氨基末端的氨基與硬脂酸羧基發(fā)生酰胺化反應(yīng)共軛連接而成,醛基化聚乙二醇則通過醛基與多肽賴氨酸殘基上的游離氨基發(fā)生反應(yīng)而生成聚乙二醇修飾的多肽-硬脂酸嫁接物(PEG modified cationic peptide stearate, PEG-STR-Pep)。以核磁共振氫譜確證兩種嫁接物的化學(xué)結(jié)構(gòu),芘熒光法測定臨界膠束濃度,透射電鏡觀察膠束的粒徑以及形態(tài)學(xué)特征,噻唑藍(lán)法考察嫁接物細(xì)胞毒性。 分別制備STR-Pep/pDNA復(fù)合物和PEG-STR-Pep/pDNA復(fù)合物,以透射電子顯微鏡觀察復(fù)合物的粒徑及形態(tài)特征,水平凝膠電泳分析嫁接物膠束與DNA的密接程度。以pEGFP-C1作為報(bào)告基因,HEK293, SKOV-3, MCF-7, Hela細(xì)胞為模型細(xì)胞,以熒光倒置顯微鏡觀察嫁接物膠束介導(dǎo)的綠色熒光蛋白表達(dá),并以流式細(xì)胞儀定量測定其轉(zhuǎn)染效率。以pGL-3質(zhì)粒為報(bào)告基因,定量考察嫁接物膠束介導(dǎo)的體外轉(zhuǎn)染水平。同時(shí),考察了STR-Pep/pDNA和PEG-STR-Pep/pDNA基因給藥系統(tǒng)的體外細(xì)胞轉(zhuǎn)染毒性。為了進(jìn)一步確證多肽中組氨酸的促內(nèi)涵體逃逸作用,以6-羧基熒光素(FAM熒光)標(biāo)記pDNA,以Lyso-Tracker標(biāo)記細(xì)胞內(nèi)內(nèi)涵體和溶酶體,觀察由STR-Pep攜載的pDNA在細(xì)胞內(nèi)實(shí)現(xiàn)內(nèi)涵體逃逸的過程。以荷SKOV-3裸鼠為模型動物,以pPEDF (plasmid of pigment epithelium-derivedfactor)為治療基因,考察STR-Pep/pPEDF基因給藥系統(tǒng)的體內(nèi)抗腫瘤藥效學(xué)。 因?yàn)镾TR-Pep嫁接物和PEG-STR-Pep嫁接物均具有顯著的膠束特性以及較好的基因轉(zhuǎn)染效率,考慮以載藥膠束與目的基因結(jié)合實(shí)現(xiàn)針對腫瘤的協(xié)同治療。本研究以堿基阿霉素(doxorubicin hydrochloride)為模型藥物,以透析法制備STR-Pep/DOX和PEG-STR-Pep/DOX載藥膠束給藥系統(tǒng)；以超濾離心-有機(jī)溶劑提取法測定載藥膠束中阿霉素的藥物含量；以透射電鏡觀察載藥膠束的形態(tài)及粒徑大�。灰許KOV-3、A549、HepG2細(xì)胞為模型細(xì)胞,評價(jià)阿霉素載藥膠束的體外藥效學(xué)。同時(shí),制備了STR-Pep/DOX/pDNA和PEG-STR-Pep/DOX/pDNA復(fù)合給藥系統(tǒng),考察其在HepG2細(xì)胞中的轉(zhuǎn)染效率。 研究結(jié)果表明,通過酰胺化反應(yīng)和親和取代反應(yīng)分別得到了STR-Pep嫁接物和PEG-STR-Pep嫁接物,核磁共振氫譜確證了STR-Pep和PEG-STR-Pep嫁接物的化學(xué)組成。STR-Pep和PEG-STR-Pep嫁接物具有良好的理化性質(zhì),臨界膠束濃度分別為182μg/mL和212μg/mL。透射電鏡觀察結(jié)果顯示STR-Pep和PEG-STR-Pep嫁接物膠束粒徑大小分別為10-20nm和50-60nm,且具有均勻的球型。STR-Pep和PEG-STR-Pep嫁接物膠束復(fù)合pDNA的能力比較強(qiáng),在水平凝膠電泳實(shí)驗(yàn)中,均可以比較低的質(zhì)量比(w/w=1, w/w=2)完全阻滯pDNA。制備的STR-Pep/pDNA和PEG-STR-Pep/pDNA基因給藥系統(tǒng)則成功實(shí)現(xiàn)了目的基因的體外轉(zhuǎn)染,STR-Pep/pDNA基因給藥系統(tǒng)在正常細(xì)胞系HEK293和腫瘤細(xì)胞系SKOV-3, MCF-7和HeLa中均達(dá)到了較高的轉(zhuǎn)染效率及轉(zhuǎn)染水平,在部分腫瘤細(xì)胞系中的轉(zhuǎn)染能力已經(jīng)非常接近于甚至超越了LipofectamineTM2000。 PEG-STR-Pep/pEGFP基因轉(zhuǎn)染系統(tǒng)則在HEK293細(xì)胞系中介導(dǎo)了明顯的綠色熒光蛋白表達(dá)。在體內(nèi)抗腫瘤藥效學(xué)研究中,STR-Pep/pDNA基因給藥系統(tǒng)取得了比較好的療效。同時(shí),STR-Pep和PEG-STR-Pep嫁接物膠束對于阿霉素具有比較高的攜載能力,包封率分別達(dá)到了54.1%和60.8%,STR-Pep/DOX/pEGFP和PEG-STR-Pep/DOX/pEGFP復(fù)合給藥系統(tǒng)在體外轉(zhuǎn)染實(shí)驗(yàn)中顯示了一定的轉(zhuǎn)染能力和發(fā)展?jié)摿?相信隨著研究的深入,將會取得較好的協(xié)同治療效果。
[Abstract]:The cationic polypeptide-stearic acid graft has a rich positive charge, can be combined with plasmid DNA (pDNA) through electrostatic interaction, thereby providing great possibility for realizing effective in-vitro gene transfection and in vivo anti-tumor treatment effect. In this study, cationic polypeptide-stearic acid/ pDNA complex and polyethylene glycol modified polypeptide-stearic acid/ pDNA complex gene administration system were constructed. Cationic polypeptide-stearic acid grafting (STR-Pep) is linked to the glycolytic reaction by amino acid at amino terminus of polypeptide and stearic acid. and the aldehyde-containing polyethylene glycol generates polyethylene glycol-modified polypeptide-stearic acid grafting (PEG-STR-Pe) through reaction between an aldehyde group and a free amino group on the polypeptide lysine residue. p). The chemical structure of the two kinds of grafts was confirmed by nuclear magnetic resonance hydrogen spectrum, the critical micelle concentration was determined by fluorescence method, the particle size and morphology of the micelles were observed by transmission electron microscope, and the graft cells were examined by means of blue-blue method. Toxicity. STR-Pep/ pDNA complex and PEG-STR-Pep/ pDNA complex were prepared respectively. The particle size and morphology of the complex were observed by transmission electron microscopy. The micelle and DNA of the graft were analyzed by horizontal gel electrophoresis. Using pEGFP-C1 as reporter gene, HEK293, SKOV-3, MCF-7 and Jurkat cells as model cells, the expression of green fluorescent protein mediated by graft micelle was observed by fluorescence inversion microscope, and quantified by flow cytometry. Its transfection efficiency is characterized by that pGL-3 plasmid is used as a reporter gene, and the micelle-mediated body of the graft is quantitatively detected. In addition, STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene were examined in vitro. In order to further confirm the escape of histidine in the polypeptide, pDNA was labeled with 6-streptavidin (FAM fluorescence), and the contents and lysosomes in the cell were labeled with Na-Tracker, and the expression of pDNA carried by STR-Pep in the cells was observed. To study the internal resistance of the STR-Pep/ pPEDF gene to the drug delivery system using the pPEDF-3 nude mouse as the model animal and pPEDF as the model animal. Tumor pharmacodynamics, because STR-Pep grafts and PEG-STR-Pep grafts both have significant micellar properties and better gene transfection efficiency, taking into account the combination of drug-carrying micelles with the target gene to implement the needle In order to prepare STR-Pep/ DOX and PEG-STR-Pep/ DOX micelle administration system by dialysis method, the drug content of doxorubicin in drug-loaded micelle was determined by ultrafiltration centrifugation-organic solvent extraction. The morphology and particle size of drug-loaded micelles were observed by transmission electron microscope. Using SKOV-3, A549 and HepG2 cells as model cells, adriamycin-loaded drug was evaluated. In vitro pharmacodynamics of micelles, STR-Pep/ DOX/ pDNA and PEG-STR-Pep/ DOX/ pDNA composite drug delivery system were prepared. The results showed that STR-Pep grafts and PEG-STR-Pep grafts were obtained by chlorosulfonation reaction and substitution reaction. STR-Pep and PEG-STR were confirmed by nuclear magnetic resonance hydrogen spectroscopy. The chemical composition of Pep grafts, STR-Pep and PEG-STR-Pep grafts have good physical and chemical properties, and the critical micelle concentration is 182. m The particle size of STR-Pep and PEG-STR-Pep was 10-20nm and 50-60, respectively. nm, and has a uniform spherical shape. STR-Pep and PEG-STR-Pep graft micelle complex pDNA has strong ability, and the low mass ratio (w/ w = 1, w/ w) can be compared in the experiment of horizontal gel electrophoresis. (2) Complete blocking of pDNA. STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene delivery systems were successfully transfected in vitro, and the STR-Pep/ pDNA gene administration system was found in normal cell line HEK293 and tumor cell line SKOV-3, MCF-7 and HeLa. High transfection efficiency and transfection level, the ability to transfect in some tumor cell lines has been very close to even more than Josephine. MineTM2000. PEG-STR-Pep/ pEGFP gene transfection system is mediated in HEK293 cell line. Significant green fluorescent protein expression. In vivo antitumor pharmacodynamic studies, STR-Pep/ pDNA gene was administered. At the same time, STR-Pep and PEG-STR-Pep graft micelles had higher carrying capacity for doxorubicin, and the entrapment efficiency reached 54. 1% and 60. 8% respectively. STR-Pep/ DOX/ pEGFP and PEG-STR-Pep/ DOX/ pEGFP composite drug delivery systems showed certain transfection ability and development potential in vitro transfection experiments. Force, believe that as the study goes deep, it will
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R94
本文編號：2311902
[Abstract]:The cationic polypeptide-stearic acid graft has a rich positive charge, can be combined with plasmid DNA (pDNA) through electrostatic interaction, thereby providing great possibility for realizing effective in-vitro gene transfection and in vivo anti-tumor treatment effect. In this study, cationic polypeptide-stearic acid/ pDNA complex and polyethylene glycol modified polypeptide-stearic acid/ pDNA complex gene administration system were constructed. Cationic polypeptide-stearic acid grafting (STR-Pep) is linked to the glycolytic reaction by amino acid at amino terminus of polypeptide and stearic acid. and the aldehyde-containing polyethylene glycol generates polyethylene glycol-modified polypeptide-stearic acid grafting (PEG-STR-Pe) through reaction between an aldehyde group and a free amino group on the polypeptide lysine residue. p). The chemical structure of the two kinds of grafts was confirmed by nuclear magnetic resonance hydrogen spectrum, the critical micelle concentration was determined by fluorescence method, the particle size and morphology of the micelles were observed by transmission electron microscope, and the graft cells were examined by means of blue-blue method. Toxicity. STR-Pep/ pDNA complex and PEG-STR-Pep/ pDNA complex were prepared respectively. The particle size and morphology of the complex were observed by transmission electron microscopy. The micelle and DNA of the graft were analyzed by horizontal gel electrophoresis. Using pEGFP-C1 as reporter gene, HEK293, SKOV-3, MCF-7 and Jurkat cells as model cells, the expression of green fluorescent protein mediated by graft micelle was observed by fluorescence inversion microscope, and quantified by flow cytometry. Its transfection efficiency is characterized by that pGL-3 plasmid is used as a reporter gene, and the micelle-mediated body of the graft is quantitatively detected. In addition, STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene were examined in vitro. In order to further confirm the escape of histidine in the polypeptide, pDNA was labeled with 6-streptavidin (FAM fluorescence), and the contents and lysosomes in the cell were labeled with Na-Tracker, and the expression of pDNA carried by STR-Pep in the cells was observed. To study the internal resistance of the STR-Pep/ pPEDF gene to the drug delivery system using the pPEDF-3 nude mouse as the model animal and pPEDF as the model animal. Tumor pharmacodynamics, because STR-Pep grafts and PEG-STR-Pep grafts both have significant micellar properties and better gene transfection efficiency, taking into account the combination of drug-carrying micelles with the target gene to implement the needle In order to prepare STR-Pep/ DOX and PEG-STR-Pep/ DOX micelle administration system by dialysis method, the drug content of doxorubicin in drug-loaded micelle was determined by ultrafiltration centrifugation-organic solvent extraction. The morphology and particle size of drug-loaded micelles were observed by transmission electron microscope. Using SKOV-3, A549 and HepG2 cells as model cells, adriamycin-loaded drug was evaluated. In vitro pharmacodynamics of micelles, STR-Pep/ DOX/ pDNA and PEG-STR-Pep/ DOX/ pDNA composite drug delivery system were prepared. The results showed that STR-Pep grafts and PEG-STR-Pep grafts were obtained by chlorosulfonation reaction and substitution reaction. STR-Pep and PEG-STR were confirmed by nuclear magnetic resonance hydrogen spectroscopy. The chemical composition of Pep grafts, STR-Pep and PEG-STR-Pep grafts have good physical and chemical properties, and the critical micelle concentration is 182. m The particle size of STR-Pep and PEG-STR-Pep was 10-20nm and 50-60, respectively. nm, and has a uniform spherical shape. STR-Pep and PEG-STR-Pep graft micelle complex pDNA has strong ability, and the low mass ratio (w/ w = 1, w/ w) can be compared in the experiment of horizontal gel electrophoresis. (2) Complete blocking of pDNA. STR-Pep/ pDNA and PEG-STR-Pep/ pDNA gene delivery systems were successfully transfected in vitro, and the STR-Pep/ pDNA gene administration system was found in normal cell line HEK293 and tumor cell line SKOV-3, MCF-7 and HeLa. High transfection efficiency and transfection level, the ability to transfect in some tumor cell lines has been very close to even more than Josephine. MineTM2000. PEG-STR-Pep/ pEGFP gene transfection system is mediated in HEK293 cell line. Significant green fluorescent protein expression. In vivo antitumor pharmacodynamic studies, STR-Pep/ pDNA gene was administered. At the same time, STR-Pep and PEG-STR-Pep graft micelles had higher carrying capacity for doxorubicin, and the entrapment efficiency reached 54. 1% and 60. 8% respectively. STR-Pep/ DOX/ pEGFP and PEG-STR-Pep/ DOX/ pEGFP composite drug delivery systems showed certain transfection ability and development potential in vitro transfection experiments. Force, believe that as the study goes deep, it will
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R94
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 李超;韓金路;王玉剛;孫英凱;;流式細(xì)胞儀的工作原理及應(yīng)用[J];中國實(shí)用醫(yī)藥;2009年20期
，本文編號：2311902
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