受体介导基因转移的策略研究与进展

基因转移是基因治疗中一个关键的环节。受体介导的基因转移具有靶向、有效和安全、无毒的特点,且操作简便,因此有望成为一种理想的体内基因转移技术用于基因治疗。     

O139霍乱弧菌CTAKΦ的基因水平转移的研究

目的 探讨CTAKΦ介导的霍乱弧菌毒素基因水平转移。方法 用抗生素敏感试验筛选出几组基因水平转移试验组合的供、受体菌，以便于用抗性筛选来确定基因转移的情况。在这几组试验组合中分别用接合转移、上清水平转移、培养液水平转移的方法，检查供体菌将CTAKΦ的A^RK^R抗性转移给受体菌的能力。结果 供体菌FJ97129、DX29能通过多种转移方式，以很高的频率将CTAKΦ的A^RK^R抗性转移给受体菌HK42、XJ93172、GD3188、7763、569B和94001；而受体菌IEM101对CTAKΦ有一定的抗感染能力。结论 CTAKΦ能够介导霍乱弧菌中霍乱毒素基因的水平转移。     

体外培养淋巴细胞基因转移的实验探讨

以ＰＨＡ、ＩＬ－２体外诱导培养２～３ｗｋ的淋巴细胞作为受体细胞，在脂质体介导下进行腺苷脱氨酶及β－半乳糖苷酶基因转移。结果表明：外周血淋巴细胞在体外经ＰＨＡ、ＩＬ－２诱导可大量增殖，其中ＣＤ３＋细胞占９０％以上。脂质体介导外源性ＤＮＡ阳性转染率可高达３０％～４０％以上。转移后细胞表达有活性的基因表达产物。提示应用脂质体对体外扩增的淋巴细胞实施基因转移，其方法简便，转移率高，转移后基因在受体细胞中能较好地表达     

表皮生长因子受体介导的新型肝癌靶向性基因转移

如何使得外源基因稳定地从吞噬溶酶体中释放 ,是提高受体介导基因转移系统转移效率的关键。本文以绿色荧光蛋白质粒为报告基因 ,合成针对表皮生长因子受体 (EGFR)的相应 16肽配体寡肽和流感病毒血凝素HA2 0寡肽 ,并与多聚赖氨酸连接 ,连接物与绿色荧光蛋白报告基因按 1∶1混合 ,构建新型肝癌靶向性转移系统 ,命名为四元复合体。分别以四元复合体和脂质体在体内外进行基因转染 ,流式细胞仪和激光共聚焦显微镜检测绿色荧光蛋白表达。结果表明 ,四元复合体介导基因定向转染至肿瘤细胞 ,其转染率为 44 95 % ,显著高于脂质体转染组( 3 3 0 9% ,P <0 0 5 ) ,动物实验证明 ,四元复合体介导绿色荧光蛋白特异性表达于肿瘤细胞 ,具有良好的应用前景     

基因治疗转移方法研究进展

基因治疗已经广泛应用于遗传病、肿瘤等疾病治疗临床试验，基因转移方法是基因治疗研究的基础和关键，脂质体介导的基因转移是物理化学方法中的最佳选择；反转录病毒介导的基因转移系统较为成熟．临床运用最广泛；腺病毒，腺病毒相关病毒基因转移系统也已进入临床试验，单纯疤疹病毒、痘苗病毒、细小病毒以及哺乳动物细胞人工染色体的基因转移系统也在研究之中。     

基因治疗转移方法研究进展

基因治疗已经广泛应用于遗传病、肿瘤等疾病治疗临床试验，基因转移方法是基因治疗研究的基础和关键，脂质体介导的基因转移是物理化学方法中的最佳选择；反转录病毒介导的基因转移系统较为成熟，临床运用最广泛；腺病毒，腺病毒相关病毒基因转移系统也已进入临床试验，单纯疱疹病毒、痘菌病毒、细小病毒以及哺乳动物细胞人工染色体的基因转移系统也在研究之中。     

DNA介导Ig基因转移及表达抗人红细胞抗体活性研究

DNA介导转移基因的技术已广泛地应用于分子遗传学领域。尤其是用于分离哺乳类基因、癌基因和鉴定基因等范围,国内外已发表不少有关的研究报告。但通过DNA介导免疫球蛋白(Ig)基因转移及表达其产物方面的研究报告,国外不多,国内尚未见     

基因治疗中的受体介导反义RNA转移技术

在调节特定基因的表达上,反义RNA是一种重要的手段。但由于存在降解、非特异性转移及安全性等问题,反义RNA在个体基因治疗中无法得到充分利用。受体介导的内吞作用为定向转移反义RNA提供了一种运载工具,它可以将反义RNA定向、高效、低毒、高安全性地转移到靶细胞中发挥作用.因而将大大加快反义RNA基因治疗的临床应用。     

趋化因子及其受体的功能

趋化因子是一类控制细胞定向迁移的细胞因子。其功能行使由趋化因子受体介导。通过基因敲除、抗体封闭及转基因技术已证明 ,趋化因子系统在病原体的清除、炎症反应、病原体感染、细胞及器官的发育、创伤的修复、肿瘤的形成及其转移、移植免疫排斥等方面都起着重要的作用。本文综述了趋化因子及其受体的生物学功能的研究进展     

一种通过表皮生长因子受体介导的基因转移系统

目的发展一种针对表皮生长因子过量表达的肿瘤细胞的基因转移系统。方法人工合成了一种Ｎ端含多个赖氨酸残基，Ｃ端为人表皮生长因子（ＥＧＦ）的受体结合域的３３肽，此合成肽（Ｋ９Ｅ肽）既具有ＤＮＡ结合活性，又能为细胞表面ＥＧＦ受体所识别并内在化。Ｋ９Ｅ肽与萤火虫荧光素酶表达质粒ｐＥＢｌｕｃ按一定比例结合形成核酸复合物，以此为基础，利用受体介导的基因转移技术，组建了外源基因转移系统。结果核酸复合物直接加入于类上皮癌细胞Ａ４３１的培养液中，４８小时后可从细胞裂解液中测出荧光素酶的显著表达。结论该系统可特异性地将外源基因导入ＥＧＦ受体过量表达的肿瘤细胞。     

灭活柯萨奇B1病毒在原代培养骨骼肌细胞中促进基因转移 …

研究灭活后的柯萨奇病毒在原代培养骨骼肌细胞中，对报道基因ｐＣＤＮＡ３－ＬａｃＺ转移效率的影响。方法用聚乙二醇沉淀和蔗糖低温超速离心法提纯并用β－丙内酯灭活病毒，将灭活ＣＶＢ１，转铁蛋白聚赖氨和报道基因设计成不同浓度组合，观察肌肉细胞中β－乳糖苷酶染色阳性的细胞数。     

Plasmid DNA-based gene transfer with ultrasound and microbubbles

Gene therapy offers a novel approach for the prevention and treatment of a variety of diseases, but it is not yet a common option in the real world because of various problems. Viral vectors show high efficiency of gene transfer, but they have some problems with toxicity and immunity. On the other hand, plasmid DNA-based gene transfer is very safe, but its efficiency is relatively low. Especially, plasmid DNA gene therapy is used for cardiovascular disease because plasmid DNA transfer is possible for cardiac or skeletal muscle. Clinical angiogenic gene therapy using plasmid DNA gene transfer has been attempted in patients with peripheral artery disease, but a Phase III clinical trial did not show sufficient efficiency. Recently, a Phase III clinical trial of hepatocyte growth factor gene therapy in peripheral artery disease (PAD) showed improvement of ischemic ulcers, but it could not salvage limbs from amputation. In addition, a Phase I/II clinical study of fibroblast growth factor gene therapy in PAD extended amputation-free survival, but it seemed to fail in Phase III. In this situation, we and others have developed plasmid DNA-based gene transfer using ultrasound with microbubbles to enhance its efficiency while maintaining safety. Ultrasound-mediated gene transfer has been reported to augment the gene transfer efficiency and select the target organ using cationic microbubble phospholipids which bind negatively charged DNA. Ultrasound with microbubblesis likely to create new therapeutic options inavariety of diseases.     

重组腺病毒介导GDNF基因在多种细胞的高效转移和表达

用作者等所构建的重组腺病毒AdCMVgdnf直接感染多种细胞系和大鼠中脑原代神经元细胞，多重感染复数为20PFU／ml。采用免疫组织化学染色方法检测GDNF表达，观察所构建的GDNF重组腺病毒在各种细胞中介导GDNF基因转移和基因表达的能力。结果表明在一系列神经细胞或非神经细胞中，GDNF重组腺病毒均可介导GDNF基因的高效转移和表达。提示GDNF重组腺病毒可作为帕金森氏病基因治疗的高效基因转移载体。     

Ligand–Histone H1 Conjugates: Increased Solubility of DNA Complexes, But No Enhanced Transfection Activity

We introduced galactose and a short RGD sequence as ligands into H1 histone to target the asialoglycoprotein receptor or integrins on cells expressing these receptors. The efficiency of the gene transfer mediated by galactosylated H1 histone was strongly affected by the transfection conditions. Galactosylation of H1 led to an increase of the basic H1-mediated gene transfer activity only, when H1 itself did not develop its optimal transfection activity. Under other conditions any specific gene transfer mediated by the asialoglycoprotein receptor was covered by the high transfection efficiency of H1 itself. Similar results of a marginal increase in the transfection efficiency were obtained by conjugates of a short RGD sequence and H1. This unexpected failure in the receptor specificity of both conjugates could be due to the unspecific cell-binding capacity of the H1 moiety and to increasing solubility of the complexes as shown by gel shift and solubility measurements.     

Enhanced efficiency of lactosylated poly-L-lysine-mediated gene transfer into cystic fibrosis airway epithelial cells

Lactosylated poly-L-lysine is a nonviral vector that transfers genes into airway epithelial cells, including those from individuals with cystic fibrosis (CF). Substitution of 40% of the epsilon-amino groups of poly-L-lysine with lactosyl residues not only provided a ligand for receptor-mediated endocytosis, but also reduced the toxicity when compared with nonsubstituted poly-L-lysine. Lactosylated poly-L-lysine/pCMVLuc complex is not toxic to cells in amounts that gave the maximum gene expression. The level of gene expression was regulated by using different combinations of chloroquine, glycerol, and E5CA peptide. Using cultured CF cells, chloroquine, combined with E5CA peptide, increased the transfer of the pCMVLuc/ lactosylated poly-L-lysine complex 10,000-fold compared with transfer without additives. In many systems, a high efficiency is of paramount importance and the enhancing agents can be used to modulate the expression of the gene. For example, transfer of pCMVLacZ/lactosylated poly-L-lysine complexes with chloroquine added to the transfection medium gave only 20% transfection efficiency of the reporter gene. However, when chloroquine was combined with glycerol, the efficiency was increased to 90%, thus approaching that reported with viral vectors. This highly efficient vector may be of great value for the future development of gene transfer systems.     

Control of gene transfer on a DNA-fibronectin-apatite composite layer by the incorporation of carbonate and fluoride ions

Gene transfer techniques are useful tools for controlling cell behavior, such as proliferation and differentiation. We have recently developed an efficient area-specific gene transfer system using a DNA-fibronectin-apatite composite layer (DF-Ap layer). In this system, partial dissolution of the composite layer is likely to be a crucial step for gene transfer. In the present study, layer solubility was adjusted by incorporating various contents of carbonate or fluoride ions into the DF-Ap layer via ionic substitution for the apatite crystals. Carbonate ion incorporation increased the solubility of the DF-Ap layer, thereby increasing the efficiency of gene transfer on the layer. In contrast, the incorporation of fluoride ions decreased the solubility of the DF-Ap layer, thereby decreasing the efficiency and delaying the timing of gene transfer on the layer dose-dependently. The present gene transfer system with controllable efficiency and timing would be useful in tissue engineering applications because cell differentiation can be induced effectively by regulating appropriate gene expression with suitable timing.     

Easy stable transfection of a human cancer cell line by electrogene transfer with an Epstein–Barr virus-based plasmid vector

We report an easy and stable transfection technique using electrogene transfer with a nonviral Epstein–Barr (EB) virus-based vector. To achieve stable transfection of human breast cancer cells, we conducted electrogene transfer of an EB virus-based plasmid vector (reduced size of oriP) containing the enhanced green fluorescence protein (eGFP) gene. Because the EB virus-based vector exhibits high transfer efficiency and strong persistent transgene expression as a result of autonomous replication in human cells, and as Nucleofector electrogene transfer can achieve highly efficient gene transfection, this method is particularly suitable for generation of stably transfected cell lines.     

A defective HIV-1 vector for gene transfer to human lymphocytes

Conclusions All practical development of HIV-1 vectors for in vivo testing will require high titers of recombinant virus free of helper virus. To date, an HIV-based vector system with an efficiency of gene transfer comparable to that achieved by the Mo-MuLV-based system has not yet been developed. Such development will require a better definition of the optimal placement of viral cis-acting sequences within the HIV-1 vector. The establishment of stable packaging cell lines should increase the efficiency of production of recombinant HIV-1 viruses for future use. T-lymphocytes are, in fact, an important potential target for therapeutic gene transfer. The possible advantages of such vectors over currently available retroviral vectors suggest that the continued study of HIV-1 vectors for gene transfer to human T-lymphocytes is warranted.Abbreviations HIV Human immunodeficiency virus - LTR Long terminal repeat     

Viscoelastic gel formulations enhance airway epithelial gene transfer with viral vectors

Advances in gene transfer to the conducting airways for the treatment of pulmonary diseases such as cystic fibrosis have identified several vector classes that transduce airway epithelia in vitro and in animal models. One barrier to epithelial gene transfer is the rapid removal of materials from the airway surface via mucociliary clearance. This host defense mechanism limits gene transfer efficiency to airway epithelial cells. Here we show that formulation of gene transfer vectors with viscoelastic gels provides longer epithelial residence time and increases vector-mediated gene transfer efficiency. Gene transfer with adenoviral, adeno-associated, and lentiviral vectors all significantly improved after formulation with viscoelastic gels designed to slow mucociliary clearance. Importantly, viscoelastic gel formulations enhanced vector transduction to the conducting airways, the desired treatment target for diseases such as cystic fibrosis.     

电脉冲介导基因在小鼠杂交瘤细胞的高效转移

本文应用电脉冲介导基因转移法,获得pSV2-neo基因在小鼠杂交瘤细胞(B889)中的高效转移和稳定表达。研究了电场强度、脉冲宽度、脉冲次数等电场参数对基因转化频率的影响和小鼠杂交瘤细胞最适电转移条件。