摘要
基因治疗有三个重要环节,即目的基因、转基因载体和靶细胞。基因导入系统是基因治疗的核心技术。目前,应用于基因治疗的载体主要有病毒载体系统和非病毒载体系统。病毒载体转染效率高,是体内基因治疗的主要工具,但安全性存在隐患及有免疫原性,体内不能反复应用。而非病毒载体具有安全性高、免疫原性低、易于对DNA进行操作等优点,故人们愈来愈重视人工合成的非病毒载体的研究,而解决靶向性问题是非病毒中载体中最为关注的问题,理想的载体系统是能将治疗基因输送到并进入特定的靶细胞,从而能在该细胞中得到有效表达,这对于恶性肿瘤基因治疗尤为重要。以受体靶向的非病毒载体系统是最常用和有效的策略,利用细胞表面表达特异性的受体或蛋白,将特定的配体分子或片段与载体连接形成分子偶联体,使DNA能靶向性地转到表达受体的细胞。同时,针对非病毒载体的缺陷及DNA转移过程中的内吞小泡释放问题、转运入核问题,根据具体情况选择合适的载体,并对其作进一步的优化、改善以获得满意的治疗或应用效果。目前常用的非病毒载体包括裸DNA,脂质体载体及阳离子多聚物型载体等。聚乙烯亚胺(polyethylenimine,PEI)是最常用的阳离子多聚物非病毒载体,PEI可把质粒DNA缩合(condense)成数百纳米大小的颗粒,通过静电作用黏附到细胞表面上,被动内吞。PEI在吞噬泡内不能降解,同时保护DNA免受溶酶体降解;另外,PEI有渗透性肿胀效应,导致吞噬泡破裂,使DNA进入胞浆,并促进DNA进入细胞核。本实验利用PEI作为载体系统的骨架,同时利用FGF受体(fibroblast growth factor receptor,FGFR)和整合素在大多数肿瘤细胞和肿瘤新生血管高表达的特点,设计了能与肿瘤细胞表面相应受体结合的寡肽:碱性细胞生长因子(basic fibroblast growth factor,bFGF)寡肽、靶向于整合素的RGD寡肽,利用交联技术将寡肽与PEI偶联,构建了新型的非病毒基因载体系统,以增加其对肿瘤细胞及其新生血管的靶向性,旨在用于恶性肿瘤基因治疗的研究。
研究分两部分:第一部分,PEI转染参数的测定及转染条件的优化。第二部分,靶向于FGFR的bFGF寡肽和靶向于整合素的RGD寡肽的设计与合成,bFGF寡肽/RGD寡肽单靶向的PEI转基因载体的构建及其介导基因转移的有效性和靶向性研究;bFGF寡肽和RGD寡肽联合靶向的PEI转基因载体的制备。
习而绷眨甲巴泊.恨L月民之二自.月眨月睡冲全刁‘因月之,卜白匀月升门回
中j忆按畏.
第一部分PEI转染效率的优化
目的:用分枝状25 kDa PEI作为转基因载体,介导报告基因质粒的转染,测定转染效
率的多种影响因素,优化转染条件,为合成更复杂的载体积累数据。
方法:利用电泳阻滞实验测定PEI与DNA的结合能力。利用编码增强型绿色荧光蛋自
的pEGFP质粒和编码p一半乳糖苍酶的PSVp质粒及Pc0NA3.甲质粒作为报告基因,通过PEI
转染pEGFP质粒和psvp质粒及peDNA3.邓质粒,检测自蛋白、血清、稀释溶媒、氯喳等
对PEI转染效率的影响,探索操作方法(包括转染次数、水平摇动、转染复合物与细胞温育
的时间等)和质粒因素(质粒质量、纯度等)对转染效率的影响。通过MTT法测定PEI的
细胞毒性。
结果:经过数据推导,得出PEI与ONA的N用比=7.53xb/c,其中N为PEI中的氮含
量,P为DNA中的磷含量,b为PEI的质徽扭g),‘为质粒的质量(雌)。PEI对COS一7细
胞和NIH3T3细胞存在一定的毒性,其C50(50%死亡浓度)为7一8林g八111。电泳阻滞试验,
PEI/D NA的N用比在2.5~3.0以上可完全阻滞DNA在电泳中的迁移。通过一系列细胞的
研究,证明PEI心NA转染效率一般以N于=7.5~10最仕。生理盐溶液作为配制PEI心NA复
合物的溶媒,转染效率高于5%葡萄糖溶液。溶酶体抑制剂氯哇降低PEI转染效果而且增加
PEI细胞毒性。培养液中的自蛋自、血清显著降低转染效率。试验发现,新鲜制备的PEI心NA
复合物转染效率>4oC放置24h>>一sooC冻存24h>>一Zooe冻存24h,<0.05)。
PEI心NA转染psVp后,12h不能测到目的基因表达产物,转染后24h目的基因开始
表达,在36h表达量达峰值,并持续数大,然后表达量开始卜降。质粒中生物活性抑制剂
(质粒提取试剂盒的残留成分,内毒素等)显著降低转染效率,通过超滤除去截流分子量小
于30,000的物质,显著增加转染效率。转染效率与质粒用量呈剂量依赖效应。
结论:PEI是有效的体外真核细胞转染剂,可用于合成更复杂的转基因载体;本研究检
测优化了PEI的转染条件井应用于卜而的实验研究。
第二部分b「G「寡肤偶联的和整合素的RGD寡肤偶联的PE!载体的设计和制备
目的:设计合成新刑的bFGF寡肤靶向的PEI转基因系统、靶向于整合素的PEI转基因
载体,研究该载体复合物系统用于基闪转移的效率。许多病毒感染细胞需要双受体机制,除
细胞特异性受体外,整合素是许多病毒的第二受体,本研究模拟病毒双受体转染细胞的机制,
制备整合素与FGFR双受体介导的PEI转基因载体。同时探讨核定位信号(nuc!ear locational
signal,NLs)肤对pEI心NA转染效率的影响。
方法:根据bFGF(1 55 AA)的三维结构、bFGF分子与FGFR(fibroblast growth factor
The prospect of curing inherited and acquired diseases through gene therapy has engendered considerable effort toward the development of gene transfer vectors. Most ongoing human gene therapy protocols rely on recombinant retroviral and adenoviral vehicles, which risk encountering acute safety and immunological problems with large scale or repeated use, besides their limited carrier capacity. Synthetic vectors, although currently orders of magnitude less efficient than biological vectors, are increasingly being considered to be possible solutions as well.
Polyethylenimine (PEI) is the most common used polycation gene delivery vector, which can condense plasmids DNA into the particles of hundreds of nanometer or so. PEI/DNA particles abundant with positive charge adhere to negative charged mucoproteins on the surface of the cells, then passively pinocytized into the cells. PEI can not be decomposed by enzymes in the endosome, has a "proton sponge effect" which make endosome osmotic swelling to rupture, and help DNA escape into the cytosol from the endosome. Receptor targeted PEI can enhance PEI's transfection efficiency. In this study, the oligopeptides of basic fibroblast growth factor (bFGF) and RGD oligopeptide were coupled with PEI, aiming at integrins and fibroblast growth factor receptor (FGFR) which are highly expressee on neoplasm neovasculature.
The study was divided into two parts: 1) To determine the related parameters affecting PEI/DNA transfection and to optimize the PEI/DNA transfection efficiency; 2) To design and synthesize bFGF oligopeptide and RGD oligopeptide binding integrins, to conjugate oligopeptide with PEI, and to assay their transfection efficiency. At the same time, we investigated the effect of nuclear locational peptide (NLS) on PEI/DNA transfection efficiency. Additionally, we developed a novel bFGF oligopeptide/RGD peptide dual-targeted PEI gene delivery vectors and investigated its transfection efficiency in vitro and in vivo.
Part I : The optimization of PEI/DNA transfection efficiency
Objective: We chose branched 25 kDa PEI as plasmids DNA delivery vector. In this part, parameters related to PEI/DNA transfection were investigated and PEI/DNA transfection efficiency was optimized, and the data were used for the synthetic targeted PEI vectors.
Methods: PEI's cytotoxicity was determined by MTT method. The binding capacity of PEI and DNA was determined through electrophoresis gel shift assay. With PEI transfection of pEGFP plasmid coding enhanced green fluoroscene protein (EGFP), pSVp plasmid and pcDNA3.1β coding β-galactosidase(p-gal), the effect of influencing factors (including albumin, serum, dilution medium, endosome inhibitor-chioroquine, etc.) were determined. The manipulation methods (including transfection times, level shaking, incubation times, etc.) and plasmids factors (including the compatibility between the plasmid and the target cells, purity
and quality of plasmids, plasmids concentration, etc.) were investigated.
Results: PEI concentration above 7~8 g/ml was significantly cytotoxic. N/P molar ratio 2.5-3.0 could completely retard DNA migration in the agarose gel electrophoresis. N/P molar ratio in the range of 7.5-10 is optimal for a variety of cells, not concomitant with significant toxicity. Dilution medium of PEI/DNA influenced the transfection efficiency, and physiological solution as dilution medium excelled over 5% glucose solution. Endosome inhibitor chloroquine decreased PEI's transfection efficiency and enhanced PEI's cytotoxity. Albumin and serum in the culture medium decreased the polycation nonviral vector's transfection efficiency.
In 12 h after PEI/DNA transfection, expressed products of reporter gene could not be detected. 24 h after the transfection, the reporter gene commenced to express, culminated in 36 h, maintained several days and later the expression amounts began to lower. The transfection efficiency was improved with additional transfection, double transfection was optimal. Level shaking of the culture plates for 30 min afte
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