心血管內(nèi)靶向定位基因遞送體系—載基因支架的實(shí)驗(yàn)研究
發(fā)布時(shí)間：2018-08-02 12:04
【摘要】：經(jīng)皮腔內(nèi)冠狀動(dòng)脈成形術(shù)(Percutaneous Transluminal Coronary Angioplasty，PTCA)是冠狀動(dòng)脈硬化性心臟病的主要治療手段，但PTCA術(shù)后血管再狹窄的發(fā)生率高達(dá)15—60％，迄今仍是臨床亟待解決的難題。轉(zhuǎn)基因技術(shù)的飛速發(fā)展為血管再狹窄的基因治療奠定了基礎(chǔ)。心血管內(nèi)基因治療的成功必須依靠有效的治療基因，，安全的載體和可以把基因(和載體)遞送到血管內(nèi)靶部位的遞送體系。心血管內(nèi)基因治療的特殊性在于很難把基因?qū)Ｒ恍赃f送到血管組織而不進(jìn)入血液循環(huán)系統(tǒng)。以往的研究大多數(shù)采用球囊導(dǎo)管向血管內(nèi)灌注基因(和載體)，研究表明，灌注到血管內(nèi)的載體大部分隨血流進(jìn)入全身循環(huán)系統(tǒng)，到達(dá)病灶局部的基因很少，達(dá)不到治療效果。 血管內(nèi)支架攜帶基因有其獨(dú)特的優(yōu)勢(shì)，可以在植入支架的同時(shí)將基因遞送到心血管內(nèi)的病灶部位，借助支架與血管壁的緊密接觸，使基因被局部血管組織吸收，隨著基因的緩慢釋放，達(dá)到長(zhǎng)期的基因轉(zhuǎn)染和表達(dá)。基因載體分為病毒載體和非病毒載體。病毒載體轉(zhuǎn)染效率高，但安全性方面存在潛在的危險(xiǎn)性。非病毒載體安全性好，易于制備，近年來(lái)倍受關(guān)注。陽(yáng)離子脂質(zhì)體和殼聚糖是目前研究最廣泛的非病毒載體，顯示出了一定的優(yōu)越性。 本課題針對(duì)心血管基因治療的主要技術(shù)難題，采用血管支架為基因遞送平臺(tái)，將抗DNA抗體—質(zhì)粒DNA—陽(yáng)離子脂質(zhì)體三元復(fù)合納米基因載體和殼聚糖基因納米粒兩種非病毒載體結(jié)合在支架上，并通過(guò)體內(nèi)外試驗(yàn)驗(yàn)證這一新型血管內(nèi)基因遞送體系的有效性和可行性。 本文第一章對(duì)PTCA術(shù)后再狹窄的發(fā)病機(jī)理及治療現(xiàn)狀進(jìn)行了綜述。 本文具體研究?jī)?nèi)容如下： 1．制備了新型抗DNA抗體—質(zhì)粒DNA—陽(yáng)離子脂質(zhì)體三元復(fù)合納米基因載體(DAC)，篩選出了最佳配方，并初步進(jìn)行了細(xì)胞攝取及細(xì)胞基因轉(zhuǎn)染實(shí)驗(yàn)。結(jié)果發(fā)現(xiàn)，抗DNA抗體—質(zhì)粒DNA—陽(yáng)離子脂質(zhì)體可以自組裝為360nm左右的球形粒子，與傳統(tǒng)的質(zhì)粒DNA—陽(yáng)離子脂質(zhì)體二元基因載體(DC組)相比，DAC體系對(duì)質(zhì)粒DNA(28.5±1.3％vs13.6±0.9％，p＜0.01)的包封率明顯增加，并且明顯地提高了細(xì)胞基因轉(zhuǎn)染效率(41.8％vs10.2％，p＜0.01)。采用雙重?zé)晒鈽?biāo)記聯(lián)合共聚焦顯微觀察發(fā)現(xiàn)抗DNA抗體可促進(jìn)質(zhì)粒DNA進(jìn)入細(xì)胞核。使用膠原對(duì)金屬支架表面進(jìn)行涂層，通過(guò)化學(xué)和免疫雙重偶聯(lián)的方法將上述DAC載體固定化在支架表面。使用放射性同位素分別標(biāo)記抗DNA抗體和質(zhì)粒DNA來(lái)測(cè)定支架結(jié)合量及釋放曲線。用細(xì)胞培養(yǎng)及動(dòng)物體內(nèi)植入實(shí)驗(yàn)驗(yàn)證了這一新型基因遞送體系的轉(zhuǎn)基因效果。結(jié)果顯示，支架表面通過(guò)化學(xué)交聯(lián)的膠原涂層具有很好的均一性
[Abstract]:Percutaneous transluminal coronary angioplasty (Percutaneous Transluminal Coronary) is the main treatment for coronary sclerosing heart disease, but the incidence of restenosis after PTCA is as high as 15-60, which is still a difficult problem to be solved. The rapid development of transgenic technology has laid the foundation for gene therapy of vascular restenosis. The success of gene therapy in cardiovascular system must depend on effective therapeutic genes, safe vectors and delivery systems that can deliver genes (and vectors) to target sites in blood vessels. The particularity of gene therapy in cardiovascular system is that it is difficult to transfer gene specifically to vascular tissue without entering the circulatory system. Previous studies have mostly used balloon catheters to infuse genes (and vectors) into blood vessels. It has been shown that most of the carriers infused into the blood vessels enter the systemic circulatory system with the blood flow, and there are few genes to the local focus, which can not achieve the therapeutic effect. Endovascular stents carry genes with unique advantages. They can be delivered to the focus of the cardiovascular system while stents are implanted, and the genes can be absorbed by the local vascular tissues by the close contact between the stents and the vascular walls. With the slow release of genes, long-term gene transfection and expression are achieved. Gene vectors are divided into viral vectors and non-viral vectors. The transfection efficiency of virus vector is high, but there is potential danger in safety. Non-viral vectors are safe and easy to be prepared, and have attracted much attention in recent years. Cationic liposomes and chitosan are the most widely studied non-viral carriers, showing some advantages. In view of the main technical problems of cardiovascular gene therapy, the vascular stent is used as the gene delivery platform. DNA antibody plasmid DNA-cationic liposome ternary composite nano-gene vector and chitosan gene nanoparticles were combined on the scaffold. The validity and feasibility of this new intravascular gene delivery system were verified by in vivo and in vitro experiments. The first chapter reviews the pathogenesis and treatment of restenosis after PTCA. The main contents of this paper are as follows: 1. A novel DNA antibody plasmid DNA-cationic liposome ternary nano-gene vector (DAC), was prepared. The best formula was selected, Cell uptake and gene transfection were also studied. The results showed that DNA antibody plasmid DNA-cationic liposomes could self-assemble into spherical particles about 360nm. Compared with the traditional plasmid DNA-cationic liposome binary gene vector (DC group), the encapsulation efficiency of plasmid DNA (28.5 鹵1.3%vs13.6 鹵0.9p < 0. 01) was significantly increased, and the efficiency of gene transfection was significantly improved (41.8 vs 10. 2% P < 0. 01). Double fluorescent labeling and confocal microscopy showed that anti DNA antibody could promote plasmid DNA into nucleus. The metal scaffold was coated with collagen and immobilized on the scaffold by chemical and immune coupling. Radioisotopes were used to label anti DNA antibody and plasmid DNA respectively to determine the binding capacity and release curve of the scaffold. The transgenic effect of this novel gene delivery system was verified by cell culture and animal implantation in vivo. The results showed that the surface of the scaffold was well homogenized by chemically crosslinked collagen coating.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R346
本文編號(hào)：2159374
[Abstract]:Percutaneous transluminal coronary angioplasty (Percutaneous Transluminal Coronary) is the main treatment for coronary sclerosing heart disease, but the incidence of restenosis after PTCA is as high as 15-60, which is still a difficult problem to be solved. The rapid development of transgenic technology has laid the foundation for gene therapy of vascular restenosis. The success of gene therapy in cardiovascular system must depend on effective therapeutic genes, safe vectors and delivery systems that can deliver genes (and vectors) to target sites in blood vessels. The particularity of gene therapy in cardiovascular system is that it is difficult to transfer gene specifically to vascular tissue without entering the circulatory system. Previous studies have mostly used balloon catheters to infuse genes (and vectors) into blood vessels. It has been shown that most of the carriers infused into the blood vessels enter the systemic circulatory system with the blood flow, and there are few genes to the local focus, which can not achieve the therapeutic effect. Endovascular stents carry genes with unique advantages. They can be delivered to the focus of the cardiovascular system while stents are implanted, and the genes can be absorbed by the local vascular tissues by the close contact between the stents and the vascular walls. With the slow release of genes, long-term gene transfection and expression are achieved. Gene vectors are divided into viral vectors and non-viral vectors. The transfection efficiency of virus vector is high, but there is potential danger in safety. Non-viral vectors are safe and easy to be prepared, and have attracted much attention in recent years. Cationic liposomes and chitosan are the most widely studied non-viral carriers, showing some advantages. In view of the main technical problems of cardiovascular gene therapy, the vascular stent is used as the gene delivery platform. DNA antibody plasmid DNA-cationic liposome ternary composite nano-gene vector and chitosan gene nanoparticles were combined on the scaffold. The validity and feasibility of this new intravascular gene delivery system were verified by in vivo and in vitro experiments. The first chapter reviews the pathogenesis and treatment of restenosis after PTCA. The main contents of this paper are as follows: 1. A novel DNA antibody plasmid DNA-cationic liposome ternary nano-gene vector (DAC), was prepared. The best formula was selected, Cell uptake and gene transfection were also studied. The results showed that DNA antibody plasmid DNA-cationic liposomes could self-assemble into spherical particles about 360nm. Compared with the traditional plasmid DNA-cationic liposome binary gene vector (DC group), the encapsulation efficiency of plasmid DNA (28.5 鹵1.3%vs13.6 鹵0.9p < 0. 01) was significantly increased, and the efficiency of gene transfection was significantly improved (41.8 vs 10. 2% P < 0. 01). Double fluorescent labeling and confocal microscopy showed that anti DNA antibody could promote plasmid DNA into nucleus. The metal scaffold was coated with collagen and immobilized on the scaffold by chemical and immune coupling. Radioisotopes were used to label anti DNA antibody and plasmid DNA respectively to determine the binding capacity and release curve of the scaffold. The transgenic effect of this novel gene delivery system was verified by cell culture and animal implantation in vivo. The results showed that the surface of the scaffold was well homogenized by chemically crosslinked collagen coating.
【學(xué)位授予單位】：中國(guó)協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類(lèi)號(hào)】：R346
【參考文獻(xiàn)】
相關(guān)期刊論文 前5條
1 錢(qián)杰,高潤(rùn)霖,史瑞文,宋來(lái)鳳,祁哲,李永利,魏英杰,孟亮,袁衛(wèi)民,司文學(xué),湯健;金屬蛋白涂層支架用于小型豬冠狀動(dòng)脈質(zhì)粒介導(dǎo)下轉(zhuǎn)基因研究[J];中國(guó)循環(huán)雜志;1999年03期
2 戴軍,高潤(rùn)霖,湯健,宋來(lái)鳳,魏英杰,宋莉,李永利,唐承君,史瑞文;蛋白涂層支架攜帶一氧化氮合酶基因轉(zhuǎn)染小型豬冠狀動(dòng)脈的可行性研究[J];中國(guó)循環(huán)雜志;2001年01期
3 錢(qián)杰,高潤(rùn)霖,湯健,史瑞文,宋來(lái)鳳,李永利,魏英杰,孟亮,袁衛(wèi)民,司文學(xué);質(zhì)粒介導(dǎo)下轉(zhuǎn)染血管內(nèi)皮生長(zhǎng)因子基因不能防治小型豬冠狀動(dòng)脈再狹窄[J];中國(guó)循環(huán)雜志;2002年05期
4 袁晉青,高潤(rùn)霖,史瑞文,宋來(lái)風(fēng),湯健,李永利,唐承君,陳在嘉;在小型豬模型由蛋白涂層金屬支架局部轉(zhuǎn)染尿激酶前體基因?qū)跔顒?dòng)脈再狹窄的影響[J];中華心血管病雜志;1999年05期
5 吳隱雄,Thomas Johnson,Christian Herdeg,Andreas Baumbach,Andrew C Newby,Karl K Karsch,Martin Oberhoff;包裹支架轉(zhuǎn)移治療基因到豬冠狀動(dòng)脈的實(shí)驗(yàn)研究[J];中山大學(xué)學(xué)報(bào)(醫(yī)學(xué)科學(xué)版);2003年05期
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