"流体力学转染"——安全高效的动物活体器官基因转染方法

作为一种新的简便而高效的动物活体基因转染方法,“流体力学转染”技术正日益成为生物遗传学家们进行基因研究的重要工具。该技术利用“流体力学”原理,通过裸质粒溶液的静脉注射,实现了活体肝靶向性的基因转染。“流体力学”注射后所造成的肝窗扩大和肝细胞膜通透性的增加,可能是肝内基因有效转染的重要机制。目前“流体力学方法”在基因研究的各个领域都得到了广泛的应用,文章重点介绍了该方法在基因功能、疾病基因治疗和获得基因产物等几个方面研究中的应用。同时也简要介绍了增强外源基因在靶器官内表达的进展。     

基因转染技术在肾脏病研究中的应用

基因转染技术在肾脏病机理研究及其防治中的应用已受到关注,并提出了针对肾小球、肾小管和肾间质的各种基因转染方法.成功地应用病毒载体和非病毒载体进行直接体内基因转染,并通过系膜细胞载体和小管上皮细胞载体系统进行间接体内基因转染,并获得较好效果,为研究肾脏病的发病机理和治疗方法奠定了基础.     

逆转录病毒介导的基因转染造血细胞的方法学进展

在逆转录病毒载体介导的转基因治疗造血系统疾病的研究中，如何提高病毒转染造血干细胞的效率，是基因转移技术的关键。本文对影响转染效率的因素进行了分析；并比较了不同的转染方法和条件下，逆转录病毒转染造血干细胞的效果。     

血小板衍化生长因子A基因转染成骨细胞生长特性的影响

背景：采用基因转染技术加强或改造种子细胞的功能是当今组织工程研究中的热点问题,用血小板衍化生长因子A转染成骨细胞用于牙周组织再生少见报道。 目的：观察血小板衍化生长因子A基因转染成骨细胞后对其生长特性的影响。 方法：构建血小板衍化生长因子A真核表达质粒,分离培养3T3细胞并进行不同方式的转染, RT-PCR检测血小板衍化生长因子A基因在成骨细胞中的表达;MTT法检测细胞的增殖活性;体外细胞划痕实验观察细胞的迁移情况。 结果与结论：RT-PCR检测结果显示,血小板衍化生长因子A真核表达质粒转染的细胞表达量明显增加、生长、迁移速度明显增快(P < 0.05)。说明脂质体介导血小板衍化生长因子A基因成功转染成骨细胞,并能促进细胞的增殖和迁移,为血小板衍化生长因子A基因在牙周组织中的治疗奠定基础。     

WT1基因异构体的转导及永久表达白血病细胞株的建立

目的：将携带WT1基因的4种异构体全长cDNA真核表达载体转导白血病细胞株NB4，为进一步研究WT1基因及其异构体对白血病细胞生物学功能的影响奠定实验基础。 方法： 用电穿孔的方法将携带WT1基因四种异构体全长cDNA的真核表达载体转染白血病细胞株NB4，通过药物筛选获得永久表达细胞株，并将此基因修饰细胞进行单克隆化，通过基因组DNA PCR，RT-PCR及Western印迹法证实外源基因在NB4细胞中的稳定整合及表达。 结果： WT1基因异构体成功转染白血病细胞株NB4，外源基因在NB4细胞中的稳定整合及表达。 结论： WT1基因异构体能够通过电穿孔的方法转染白血病细胞并建立永久表达细胞株。     

转染CRKL蛋白对肺癌细胞系增殖的研究

目的研究CRKL蛋白对肺癌细胞系增殖的影响及其可能机制,探讨CRKL基因在肺癌发病中的作用。方法在转染CRKL的HBE和H1299两种肺癌细胞系中,采用Western blot,流式细胞技术(FCM),集落形成实验和MTT方法检测转染CRKL后,对肺癌细胞增殖的影响。结果转染CRKL基因后,可明显上调Cyclin D1和Cyclin B蛋白在HBE和H1299两种肺癌细胞表达,增加Rb的磷酸化,促进肺癌细胞的增殖、克隆形成能力。结论 CRKL通过调节细胞周期促进肺癌细胞的增殖能力,CRKL可作为非小细胞肺癌的标记物,并可能成为治疗该肿瘤的新靶点。     

RNA干扰致基因表达沉默细胞模型的建立

目的探讨siRNA和shRNA在基因功能研究中的应用。方法用化学方法合成siRNA,以及用常规的分子克隆方法构建shRNA表达载体。以人类K562细胞为实验对象,采用lipofectamine将MDM2基因的siRNA和shRAN表达载体导入细胞后,用多重RT鄄PCR和Westernblot检测目标基因MDM2的表达水平。结果K562细胞瞬时转染MDM2siRNA48h后,MDM2蛋白质表达下降超过60%;稳定转染shRNA表达载体后,MDM2基因在mRNA和蛋白质表达水平下降超过70%。实验结果表明成功地建立了MDM2表达下调的实验细胞模型。结论siRNA和shRNA都能有效地导致目标基因表达沉默,在基因功能研究中瞬时转染和稳定转染需要配合使用以获得更理想的实验结果。     

脂质体介导哺乳动物细胞转染的改良方法

目的：提高脂质体介导的细胞转染效率.方法：在传统的脂质体生化转染过程中,介入了简单的物理转染技术,并应用有限稀释克隆技术和共聚焦荧光显微镜检测技术,对转染细胞进行了早期克隆筛选,用流式细胞术对转染细胞表达GFP的阳性率进行检测.结果：流式细胞术检测显示,通过改良法获得转染pcDNA3-GFP-PSA质粒的B16、RM1和EL4细胞,表达报告蛋白GFP的阳性百分率（26%、24%和40%）高于传统法转染的细胞（16%、16%和18%）.改良法转染的B16、RM1和EL4经过克隆筛选、液氮冻存、水浴复苏和体外连续培养2周后,外源基因报告蛋白GFP表达的阳性百分率分别为77%、69%和83%.在转染流程上,通过改良法获得单克隆转染细胞株的时间明显缩短.结论：改良的脂质体转染方法结合细胞克隆技术,能在最短时间内获得高表达外源基因的转染细胞株.     

血管内皮生长因子反义基因转染对高转移人巨细胞肺癌细胞系生 …

目的 探讨反义血管内皮生长因子（ＶＥＧＦ）基因转染在抑制恶性肿瘤生长和转移的抗肿瘤血管基因治疗中的意义。方法 利用基因重组技术构建正义和反义ＶＥＧＦ１２１ ｃＤＮＡ真核表达载体,用脂质体法转染高转移性人巨细胞肺癌细胞（ＰＧ）,经Ｎｏｒｔｈｅｍ杂交和Ｗｅｓｔｅｒｎ印迹免疫化学检测ＶＥＧＦ ｍＲＮＡ和蛋白质的表达水平,并对转染前后细胞进行体外生长和裸鼠体内生长转移等多项生物学行为实验,结果 转染反义转     

转染重组bcl-2基因缓解RA介导NTCD的电镜观察

神经胚形成 ( neurulation)中最关键的形态学变化是神经管的形成 ,在此过程中 ,正常 PCD是不可缺少的因素之一。在神经管关闭过程中发生率很高的各种神经管关闭缺陷 ( neuraltube closure defect,NTCD) ,大多数亦与凋亡异常有关。研究发现 PCD广泛存在于发育中的神经系统 ,其意义在于对神经系统进行雕塑 ,最终使人神经系统的发育达到结构的高度精细和功能的尽善尽美。但目前关于早期神经管发育中的 PCD及与原癌基因 bcl-2的关系研究极少。为此本实验用电镜技术结合全胚胎培养技术和重组基因转染技术研究转染 bcl- 2基因能否减轻 PA介…     

Efficient transfection method for primary cells

Transfection of primary cells and stem cells is a problem in the laboratory routine and further in tissue engineering and gene therapy. Most methods working effectively for cell lines in culture fail to transfect primary cells. Here we describe the use of the Nucleofector technology developed by amaxa biosystems. We were able to transfect primary human melanocytes, human coronary smooth muscle cells, human chondrocytes, and human mesenchymal stem cells with high efficiencies (28.9-45.3%). All primary cell types failed to be transfected satisfactorily by methods based on liposome-mediated transfection in our hands. The viability of the transfected cells varied between 11.2% and 75% in comparison to untreated cells. Only 200,000 cells per transfection sample were needed. In summary, this method presents an effective and fast mean for transfection of primary and stem cells demonstrated by four cell types which are only transfected with low efficiency by other methods.     

Photochemical Transfection: A Technology for Efficient Light-Directed Gene Delivery

Most synthetic gene delivery vectors are taken up in the cell by endocytosis, and inefficient escape of the transgene from endocytic vesicles often is a major barrier for gene transfer by such vectors. To improve endosomal release we have developed a new technology, named photochemical internalization (PCI). PCI is based on photochemical reactions initiated by photosensitizing compounds localized in endocytic vesicles, inducing rupture of these vesicles upon light exposure. PCI constitutes an efficient light-inducible gene transfer method in vivo, which potentially can be developed into a site-specific method for gene delivery in in vivo gene therapy. In this paper the principle behind the PCI technology and the effect of PCI on transfection with different synthetic gene delivery vectors are reviewed. PCI treatment by the photosensitizer aluminum phthalocyanine (AlPcS_2a ) strongly improves transfection mediated by cationic polymers (e.g., poly-L-lysine and polyethylenimine), while the effect on transfection with cationic lipids is more variable. The timing of the light treatment relative to the transfection period was also important, indicating that release of the DNA from early endosomes is important for the outcome of PCI-induced transfection. The possibilities of using PCI as a technology for efficient, site-specific gene delivery in in vivo gene therapy is discussed.     

Electroporation gene therapy: new developments in vivo and in vitro

Electroporation-based gene therapy has become a "hot field" for non-viral gene delivery. This review summarizes the progress made in intramuscular and intratumoral electrogenetransfer, which include new applications and modifications. The progress in dendritic cell (DC) and stem cell transfection by use of electroporation has also been discussed. Rapid progress during the past two years clearly demonstrates the great potential of this technology, but there are challenges faced by both in vitro and in vivo applications, which include how to enhance the transfection efficiency for intratumoral delivery, how to extend the duration of gene expression for intramuscular injection, and how to increase the survival rate for in vitro cell transfection. Resolving these issues will shed new light on this field.     

Rapid and highly efficient gene transfer into natural killer cells by nucleofection

Natural killer (NK) cells are important mediators of virus- and tumor-specific immune responses. The transfection of genes into NK cells has been proven difficult and so far requires infection with virus-based vectors. Here, the application of a novel nonviral, electroporation-based gene transfer method is described for the rapid and highly efficient transient transfection of NK cell lines as well as freshly isolated NK cells. In contrast to conventional methods, this technique, termed nucleofection, leads to direct transfer of DNA into the nucleus. Using reporter proteins H-2K(k), luciferase+, and enhanced yellow green fluorescent protein (EYFP) as independent read-out systems, transfection efficiencies of well over 50% were achieved in transient transfection assays. The highest luciferase activity could be measured only 4 h after transfection, whereas EYFP, when analyzed by flow cytometry, showed expression peaks after 28 h. Interestingly, best transfection efficiencies were achieved with non-dividing NK cells. The novel nuclear gene transfer method presented here is highly useful for the analysis of NK cell-specific gene regulation and should facilitate the development of NK cell-based gene therapy approaches.     

Light directed gene transfer by photochemical internalisation

Numerous gene therapy vectors, both viral and non-viral, are taken into the cell by endocytosis, and for efficient gene delivery the therapeutic genes carried by such vectors have to escape from endocytic vesicles so that the genes can further be translocated to the nucleus. Since endosomal escape is often an inefficient process, release of the transgene from endosomes represents one of the most important barriers for gene transfer by many such vectors. To improve endosomal escape we have developed a new technology, named photochemical internalisation (PCI). In this technology photochemical reactions are initiated by photosensitising compounds localised in endocytic vesicles, inducing rupture of these vesicles upon light exposure. The technology constitutes an efficient light-inducible gene transfer method in vitro, where light-induced increases in transfection or viral transduction of more than 100 and 30 times can be observed, respectively. The method can potentially be developed into a site-specific method for gene delivery in vivo. This article will review the background for the PCI technology, and several aspects of PCI induced gene delivery with synthetic and viral vectors will be discussed. Among these are: (i) The efficiency of the technology with different gene therapy vectors; (ii) use of PCI with targeted vectors; (iii) the timing of DNA delivery relative to the photochemical treatment. The prospects of using the technology for site-specific gene delivery in vivo will be thoroughly discussed, with special emphasis on the possibilities for clinical use. In this context our in vivo experience with the PCI technology as well as the clinical experience with photodynamic therapy will be treated, as this is highly relevant for the clinical use of PCI-mediated gene delivery. The use of photochemical treatments as a tool for understanding the more general mechanisms of transfection will also be discussed.     

A reverse transfection technology to genetically engineer adult stem cells

A new non-viral method of gene transfection was designed to enhance the level of gene expression for rat mesenchymal stem cells (MSCs). Pullulan was cationized using chemical introduction of spermine to prepare cationized pullulan of non-viral carrier (spermine-pullulan). The spermine-pullulan was complexed with a plasmid deoxyribonucleic acid (DNA) of luciferase and coated on the surface of culture substrate together with Pronectin of artificial cell adhesion protein. MSCs were cultured and transfected on the complex-coated substrate (reverse transfection), and the level and duration of gene expression were compared with those of MSCs transfected by culturing in the medium containing the plasmid DNA-spermine-pullulan complex (conventional method). The reverse transfection method enhanced and prolonged gene expression significantly more than did the conventional method. The reverse method permitted the transfection culture of MSCs in the presence of serum, in contrast to the conventional method, which gave cells a good culture condition to lower cytotoxicity. The reverse transfection was carried out for a non-woven fabric of polyethylene terephthalate (PET) coated with the complex and Pronectin using agitation and stirring culture methods. The two methods enhanced the level and duration of gene expression for MSCs significantly more than did the static method. It is possible that medium circulation improves the culture conditions of cells in terms of oxygen and nutrition supply and waste excretion, resulting in enhanced gene expression.     

Experimental gene therapy using p21Waf1 gene for esophageal squamous cell carcinoma by gene gun technology

In our previous study, the proliferation rate of esophageal squamous cell carcinoma cell lines, which poorly expressed p21Waf1, was found to be regulated by p21Waf1 gene transfection using adenovirus vector. In the present study, in order to examine the effect of p21Waf1 gene therapy in esophageal cancer, we used gene gun technology, which proved to be a powerful method to introduce the p21Waf1 gene into esophageal cancer cells. p21Waf1 transfection to KE3 and YES2 cells (weakly expressed p21Waf1 protein cells) showed a high expression of p21Waf1 protein after applying this gene gun technique. In KE3 and YES2 cells, statistical significant growth inhibition was observed after p21Waf1 transfection compared with LacZ transfection (KE3, p=0.0009; YES2, p<0.0001). In in vivo transfection experiments, on day 14, the estimated volume of KE3 tumors subjected to p21Waf1 gene transfection was 95% in comparison with the pretreatment volume on day 0, while the volume of KE3 tumors subjected to LacZ gene therapy increased to 268%. On day 14, the estimated volume of YES2 tumors subjected to either p21Waf1 or LacZ gene therapy increased to 474 and 686%, respectively. In KE3 and YES2 cells, significant growth inhibition was observed after combination therapy using p21Waf1 transfection and anticancer drug 5-fluorouracil (5Fu) compared with 5Fu alone (KE3, p<0.0001; YES2, p<0.0001). In conclusion, p21Waf1 gene therapy using the gene gun technique significantly inhibited the low basal p21Waf1 expressed esophageal cancer cell growth in vitro and in vivo. Furthermore, p21Waf1 transfection strongly enhanced the effect of 5Fu suggesting that p21Waf1 may prove beneficial in chemotherapy combined with gene therapy using gene gun technology in patients with esophageal cancer who have a low level of p21Waf1 expressed tumor.     

FTIR对TFAR19基因促红白血病MEL细胞凋亡作用的研究

采用 FTIR方法 ,研究了 TFAR19基因转染前后 ,小鼠红白血病 MEL 细胞内重要组分的变化。经脂质体介导基因转染 ,获得了稳定携带 TFAR19基因的 MEL- TF19细胞 ,RT- PCR检测表明 ,该基因在 m RNA水平有表达 ;撤除血清诱导凋亡 ,在此过程中进行了 FTIR检测 ,结果显示 :转染 TFAR19基因后 ,细胞内蛋白质相对核酸的含量增高 ,细胞转录活性增强 ,细胞内磷脂的相对含量降低。所有这些变化都反映了 TFAR19基因的促凋亡作用 ,并可能是前期工作中发现的细胞流变学特性发生变化的基础     

In vitro and in vivo transfection of primary phagocytes via microbubble-mediated intraphagosomal sonoporation

The professional phagocytes, such as macrophages and dendritic cells, are the subject of numerous research efforts in immunology and cell biology. The use of primary phagocytes in these investigations however, are limited by their inherent resistance to transfection with DNA constructs. As a result, the use of phagocyte-like immortalized cell lines is widespread. While these cell lines are transfection permissive, they are generally regarded as poor biological substitutes for primary phagocytes. By exploiting the phagocytic machinery of primary phagocytes, we developed a non-viral method of DNA transfection of macrophages that employs intraphagosomal sonoporation mediated by internalized lipid-based microbubbles. This approach enables the transfection of primary phagocytes in vitro, with a modest, but reliable efficiency. Furthermore, this methodology was readily adapted to transfect murine peritoneal macrophages in vivo. This technology has immediate application to current research efforts and has potential for use in gene therapy and vaccination strategies.