新型非病毒载体聚乙烯亚胺体内应用的研究进展

聚乙烯亚胺是1995年发现的重要的真核细胞基因转染载体，在非病毒载体的研究中处于重要的地位。聚乙烯亚胺体外研究已取得显著的进展，但在体内基因治疗的应用面临着一系列局限。本文综述了非病毒载体聚丙烯亚胺体内应用的障碍和克服办法，包括聚乙烯亚胺-DNA复合物的脂质体包裹或聚乙二醇化修饰等。     

聚乙烯亚胺介导基因转染的影响因素研究

目的研究非病毒基因载体聚乙烯亚胺（PEI）介导基因转染的影响因素。方法采用透射电镜观察PEI与DNA形成复合物的形态,分别考察质粒量、复合物形成时间、复合物在细胞上作用时间和氮磷比（N/P）对转染效率的影响,以筛选较优的转染条件。结果PEI可有效地与DNA形成复合物,当质粒量为每孔2μg、复合物形成时间为30min、复合物在细胞上的作用时间为4h且N/P为20时转染效率最高。结论PEI可作为合成新型非病毒载体的骨架,但必须控制有关因素才能得到最佳的和可重复的转染结果。     

壳寡糖作为基因载体的体外评价

摘要目的评价壳寡糖作为基因载体的理化性质及转染293T细胞的活性。方法选用相对分子质量小的壳寡糖,使用噻唑蓝（MTT）法检测不同浓度壳寡糖的细胞毒性,琼脂糖凝胶电泳检测壳寡糖结合及保护质粒DNA的作用,荧光显微镜观测转染结果。结果高浓度壳寡糖第2天时具有较低毒性,第4天时细胞存活率高于不做处理的对照细胞;质量比＞20倍时,壳寡糖几乎完全结合DNA,并且具有一定保护的作用;转染293T细胞后,荧光显微镜能够观测到表达的绿色荧光蛋白。结论壳寡糖可以作为基因载体转染293T细胞。     

非病毒型载体聚乙烯亚胺在基因递送应用中的研究进展

非病毒型基因载体因具有低毒性、低免疫原性及易制备等诸多优点,近年来成为制备高效低毒型基因递送载体的研究热点。聚乙烯亚胺(PEI)作为典型的非病毒型基因载体以其较好的基因压缩能力及溶酶体逃逸能力引起了广泛关注。本文对基于PEI类基因载体的体内递送过程及所面临的问题(如生物相容性低、细胞毒性、缺乏特异靶向性和目的基因释放不足等)进行了归纳与分析,并总结了相应的改进方案。此外,对基于PEI的智能响应型基因载体(内源性刺激响应型、外源性刺激响应型)的研究进展进行了综述,为开发出更加安全高效的非病毒型基因载体提供参考。     

阳离子脂质材料及其在基因转染中的应用

目的通过对已报道的阳离子脂质材料的结构及其应用的综述,为该类基因转染载体的合理设计和进一步应用提供借鉴。方法对已有的各种阳离子脂质材料的结构及基因转染特性进行分析,并探讨其对基因转染的影响。结果阳离子脂质材料在基因转染中有着巨大的应用潜力,特定的脂质材料结构赋予其在基因转染中不同的效能。结论对阳离子脂质材料结构特征的综合分析,为进一步合理构建新的高效阳离子脂质体基因递送载体提供了思路。     

非病毒载体在肿瘤基因治疗领域的研究进展

随着肿瘤基因治疗领域的研究进展，临床应用逐渐增多。载体是癌症基因治疗的主要难题。当前广泛使用的病毒载体存在的安全问题越来越受到人们的重视，已经有多种非病毒载体用于肿瘤基因治疔，如：裸DNA直接注射、阳离子脂质、阳离子聚合物。研究非病毒载体的目标是：它能像靶向的合成病毒载体那样对肿瘤组织表现出高度特异性；具有很高的转染效率；潜在的安全性问题能够被控制。     

聚乙烯亚胺非病毒基因载体结构修饰研究进展

聚乙烯亚胺（PEI）作为最经典的非病毒基因载体之一,由于其高转染效率,在基因递送领域受到极大的关注,但其毒性限制了聚乙烯亚胺的应用。本文综述了对聚乙烯亚胺进行不同的结构修饰,如多糖修饰、PEG修饰和低分子聚乙烯亚胺衍生物等,以实现在不显著降低转染效率的前提下减少细胞毒性。     

PEG-b-PLL的合成及其自组装纳米基因载体的研究

目的合成两嵌段聚乙二醇-b-聚赖氨酸共聚物(PEG-b-PLL),并评价PEG-b-PLL载基因纳米复合物。方法以端氨基PEG引发Lys(z)-NCA,首先得到两嵌段共聚物PEG-b-PZLL,然后酸解去除苄氧羰基保护基团,得到了两嵌段共聚物PEG-b-PLL。通过正负电荷吸附作用自组装形成PEG-b-PLL载基因纳米复合物,考察其性质。结果制备的PEG-b-PLL载基因纳米复合物外观圆整,呈类球形,大小均匀,平均粒径为(150.3±5.5)nm,其Zeta电位为(-15.82±2.34)mV。该复合物在血浆中稳定,具有一定的抗核酸酶降解能力,且能成功的转染HepG2细胞。结论该复合物是一种制备工艺简单,性能良好,极富潜力的非病毒基因载体。     

胍基化非病毒载体研究进展

非病毒载体具有质量可控、携带能力高和免疫原性低等优点,近些年来备受关注。理想的非病毒载体在递送基因的过程中需要克服多重生物学屏障,包括血液屏障、细胞屏障和胞内转运屏障。胍基来自于精氨酸,可以与磷酸基团形成双齿氢键,在DNA压缩、细胞膜跨越和入核等过程中发挥着重要作用。对非病毒载体进行胍基化基团的修饰,有利于克服非病毒载体在递送基因的过程中面临的生理屏障,提高基因的转染效率。本文综述了非病毒载体的胍基化修饰对于提高基因递送效率方面的研究进展,为构建新的高效非病毒载体提供了思路。     

阳离子脂质体在基因转染载体中的研究进展

目前基因治疗面临的首要技术问题是基因药物的载体,用于基因治疗的载体主要分为两大类:病毒载体和非病毒载体.病毒载体可能在体内发生基因的重组或互补,因此具有较大的潜在危险,限制了它在临床基因治疗上的应用[1].非病毒载体中发展最为成熟的是阳离子脂质体(cationic liposomes,CL)载体,它具有可自然降解、无免疫原性、可重复转染等优点,迄今已有数十种阳离子脂质体被用于基因转染[2].该项技术目前已成为基因治疗的研究热点之一,现就其进展综述如下.     

基于聚乙烯亚胺的非病毒基因载体研究进展

如今,越来越多的非病毒基因载体被应用于基因传递中,聚乙烯亚胺（PEI）作为一种重要的阳离子聚合非病毒基因传递载体受到广泛的关注。针对如何克服其生物不可降解性和细胞毒性并进一步提高转染效率的问题,笔者通过文献综合介绍了一些新的策略,并具体对以PEI为基础的新型载体的合成、新型体外转染方法的建立以及体内应用等方面的研究进展进行综述。     

Cationic Cholesterol Promotes Gene Transfection Using the Nuclear Localization Signal in Protamine

Purpose. The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin). Methods. Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37°C. The cells then were washed and cultured for another 40 h in the growth medium at 37°C before luciferase assay. Results. The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine. Conclusion. Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.     

聚乙烯亚胺衍生物作为非病毒基因载体的研究进展

综述了通过结构修饰获得的各类聚乙烯亚胺（PEI）衍生物，如PEG修饰、小分子PEI共聚、连接配体或其它高分子骨架物等，及其在基因转染和细胞毒性方面的研究进展。     

PEG化聚乙烯亚胺作为非病毒基因给药载体的研究

采用不同比例的单甲氧基聚乙二醇-琥珀酰亚胺基丙酸盐（mPEG-SPA）对支链聚乙烯亚胺（PEI）进行修饰，考察了不同接枝率的mPEG-PEI与DNA复合物的粒径、ζ‘电位、包封率，及其对A549细胞的转染效率和细胞毒性。体外转染试验表明，低PEG接枝率的PEI转染效果与PEI相当，且细胞毒性大大降低。     

鱼精蛋白／DNA复合物的构建与生物性能评价

目的研究非病毒基因载体鱼精蛋白／DNA复合物的制备方法及对其细胞毒性、不同量鱼精蛋白对pDNA的结合能力、体外细胞转染率。方法不同量鱼精蛋白与DNA在室温孵育后,得到鱼精蛋白／DNA复合物;用MTT法检测鱼精蛋白对HeLa子宫颈癌细胞的毒性作用,同时与PEI进行比较;利用琼脂糖电泳实验测定不同N／P比形成复合物时对DNA的阻滞情况;用结合沉淀试验比较不同量鱼精蛋白对包裹DNA的能力的影响;在荧光显微镜下观察比较它们对BEL-7402肝癌细胞转染率的大小。结果当鱼精蛋白／DNA复合物浓度升高时,对He—La子宫颈癌细胞的毒性均为0级,而聚乙烯亚胺（PEI）浓度升高时,对细胞的毒性明显增加;鱼精蛋白与质粒DNA形成复合物时所需的N／P比为1．5：1,较PEI／DNA复合物形成时所需的N／P比2：1要小;鱼精蛋白包裹DNA的能力随N／P比增大而增强,并且包裹DNA的能力要比PEI／DNA复合物转变得快;鱼精蛋白／DNA复合物对BEL-7402肝癌细胞的转染率较PEI／DNA复合物低,但毒性较低。结论鱼精蛋白／DNA复合物是一种制备工艺简单、细胞毒性小、对pDNA包裹能力高、转染率相对较高,具有一定应用潜力的非病毒基因载体。     

Aerosol delivery of programmed cell death protein 4 using polysorbitol-based gene delivery system for lung cancer therapy

Abstract

The development of a safe and effective gene delivery system is the most challenging obstacle to the broad application of gene therapy in the clinic. In this study, we report the development of a polysorbitol-based gene delivery system as an alternative gene carrier for lung cancer therapy. The copolymer was prepared by a Michael addition reaction between sorbitol diacrylate (SD) and spermine (SPE); the SD–SPE copolymer effectively condenses with DNA on the nanoscale and protects it from nucleases. SD–SPE/DNA complexes showed excellent transfection with low toxicity both in vitro and in vivo, and aerosol delivery of SD-SPE complexes with programmed cell death protein 4 DNA significantly suppressed lung tumorigenesis in K-ras^LA1 lung cancer model mice. These results demonstrate that SD–SPE has great potential as a gene delivery system based on its excellent biocompatibility and high gene delivery efficiency for lung cancer gene therapy.     

线粒体单基因突变罕见病的基因治疗研究进展

线粒体单基因突变罕见病是一组由单个线粒体基因突变而引发的疾病,具有病情严重、诊断困难、治愈率低等特点;其中有些疾病甚至会致残致死,给家庭和社会带来了沉重的负担。目前,小分子药物在线粒体单基因突变罕见病的大多数治疗策略中仍占据主导地位,但由于小分子药物仅能缓解疾病症状,无法改变线粒体基因突变所导致的问题,因此,众多患者仍然面临无药可治的困难境地。近年来,基因组生物学、基因编辑和基因递送系统的发展给线粒体单基因突变罕见病的治疗带来了希望。以2个具有代表性的线粒体单基因突变罕见病为例,归纳了其病理特征、基因递送屏障和临床试验中使用的治疗方法,并总结了针对该类疾病已开发的基因递送系统。最后,对基因疗法治疗线粒体单基因突变罕见病所面临的挑战和应用前景进行了系统的讨论。     

Polymeric Gene Transfection on Insulin-Secreting Cells: Sulfonylurea Receptor-Mediation and Transfection Medium Effect

Purpose In vitro transfection of secreting cells is regarded as one strategy for improved cell engineering/transplantation. Insulin-secreting insulinoma cell lines or pancreatic β-cells could be genetically engineered using designed polymeric vectors which are safer than viral vectors. This study investigates the effects of the constituents in transfection media on polymeric transfection.Methods Polyplexes conjugated with sulfonylurea (SU) were evaluated under different transfection conditions for gene transfection and their effects on cytotoxicity and insulin secretion. Several components in transfection media specifically associated with the insulin secretion pathway were amino acids, vitamins, Ca²⁺ and K⁺. The interactions of the polyplexes with insulin were monitored by surface charge and particle size to monitor how insulin as a protein influences transfection.Results For an insulin-secreting cell line (RINm5F), polyplexes in Ca²⁺-containing KRH medium (Ca²⁺(+)KRH) enhanced transfection and did not cause damage to biological functions. When adding amino acids, vitamins, or K⁺ or depleting Ca²⁺ from Ca²⁺(+)KRH, poly(l-lysine)/DNA complexes showed a greater reduction in transfection than SU receptor (SUR)-targeting polyplexes (SU-polyplex). Positively charged polyplexes interacted with insulin, developing a negative surface charge, and these interactions may cause a decrease in transfection.Conclusion The findings suggest that in vitro and ex vivo polymeric transfection of insulin-secreting cells can be modulated and enhanced by adjusting the transfection conditions.     

基于多糖聚合物的非病毒基因载体的研究进展

基因治疗是通过一定的载体或手段将功能性外源基因导入受体细胞中,通过外源基因的表达产物或是通过外源基因抑制某些基因的转录或翻译,达到治疗的目的。基因载体有2种:病毒载体传递体系和非病毒载体传递体系。病毒载体传递体系因安全问题使其在临床应用中多有限制。非病毒载体有着诸多优势,但较低的转染效率限制了其在研究及临床上的应用。本文综述近年来一类基于多糖聚合物设计的非病毒载体的研究进展,并阐述其在基因治疗中的优势及应用。     

Non-viral methods for gene transfer towards osteosarcoma therapy

Osteosarcoma, a class of cancer that originates from bone, afflicts mainly young people usually in their teenage years of life. Despite surgery and chemotherapy, the outlook for sufferers is not that positive, with a third of patients with metastatic disease not surviving past the 10-year mark. Like other neoplasms, other forms of therapeutics are being evaluated, and amongst these is gene therapy. This review discusses approaches for gene therapy of osteosarcoma using cationic liposomes and polyethylenimine in vivo. The field is still in its infancy as far as osteosarcoma is concerned and much more needs to be done to test its true potential as a feasible therapeutic modality.