反义核酸的骨架修饰及其应用

反义核酸的发展经历了反义寡核苷酸、混合骨架寡核苷酸和多肽核酸等几个阶段。这3种不同类型的反义核酸均能与DNA或RNA结合,阻断目的基因的表达。3种反义核酸的结构有较大差异,各自的性质和反义作用机理也不尽相同。尽管作用机制还不十分明确,反义核酸已广泛应用于生物学和医学等领域,作为反义药物用于治疗癌症等疾病,或作为试剂研究生物大分子的功能。     

survivin反义寡核苷酸在体外诱导胰癌细胞凋亡

目的：探讨survivin反义核酸对胰腺癌细胞株Panc-1细胞凋亡的影响.方法：用脂质体瞬时转染法介导survivin反义核苷酸处理胰腺癌Panc-1细胞后,MTT试验测定转染细胞的相对存活率,RT-PCR检测survivin mRNA表达,琼脂糖凝胶电泳分析其对Panc-1细胞的凋亡诱导作用.结果：转染survivin反义核酸的Panc-1细胞增殖明显受抑制,与对照进行比较,具有显著性差异（P＜0.05）;经琼脂糖凝胶电泳,转染survivin反义核酸的Panc-1细胞可见到DNA梯形条带,而对照细胞未见到.结论：survivin反义核酸能够诱导Panc-1细胞凋亡.     

hTERT基因反义核酸对Jurkat细胞端粒酶活性影响

目的:检测hTERT基因反义核酸对Jurkat细胞端粒酶活性的影响及其机制。方法:TRAP法检测端粒酶活性,流式细胞仪检测hTERT蛋白表达,逆转录-多聚酶链式反应检测hTERTmRNA表达。结果:检测端粒酶活性,Jurkat细胞吸光度A值为0.492±0.051,hTERT反义核酸作用48hA值降为0.351±0.051,hTERT反义核酸作用72hA值降为0.238±0.024;检测hTERT蛋白表达,Jurkat细胞hTERT蛋白阳性率89.513%±3.389%,hTERT反义核酸作用48hhTERT蛋白阳性率低至77.237%±2.872%,hTERT反义核酸作用72hhTERT蛋白阳性率低至47.767%±1.326%。而hTERT正义核酸无上述作用。hTERT反义核酸作用Jurkat细胞48h、72h对hTERTmRNA表达无影响。结论:hTERT反义核酸降低Jurkat细胞端粒酶活性,机制可能是降低hTERT蛋白表达量,对hTERTmRNA无影响。     

反义核酸抑制miR-20、miR-106表达对293T细胞增殖的影响

目的:通过反义核酸技术抑制miR-20、miR-106的表达,检测对293T细胞增殖的影响。方法:应用特异性针对miR-20、miR-106的反义核酸转染293T细胞,用形态学、流式细胞技术研究反义核酸对细胞增殖的抑制作用,用定量PCR和ELISA研究miRNA和相关靶基因的表达。结果:研究发现反义核酸可有效抑制miR-20和miR-106的表达,并明显抑制293T细胞的增殖;研究还表明反义miR-106可明显上调抑癌基因Rb的表达。结论:反义核酸通过抑制miRNA表达,上调抑癌基因Rb的表达,能明显抑制293T细胞增殖。     

反义核酸技术及其在恶性黑素瘤中的应用进展

恶性黑素瘤对化疗等多种通过凋亡途径杀伤肿瘤细胞的治疗方法疗效均不理想。近年来利用反义核酸技术选择性地封闭与细胞增殖、凋亡及耐药密切相关的靶基因,在恶性黑素瘤研究中取得了较大进展,为恶性黑素瘤的治疗提供了新的途径。体内外研究结果显示反义核酸治疗可有效抑制黑素瘤细胞增殖、促进细胞凋亡及逆转细胞耐药性。特别是针对抗凋亡基因BCL-2的反义核酸已作为一种最有希望的反义核酸药物同化疗药物联合用于恶性黑素瘤Ⅲ期临床试验。     

hTERT 基因反义核酸诱导卵巢癌细胞凋亡的研究

目的 在体外实验中，探讨hTERT反义核酸诱导卵巢癌细胞发生凋亡的可能性，揭示该凋亡发生与bcl-2和bax之间的关系。方法采用TUNEL染色法，定性、定量地研究hTERT反义核酸与卵巢癌细胞凋亡的关系；通过免疫组织化学法检测凋亡相关基因bcl-2和bax的表达。结果hTERT反义核酸在体外能诱导卵巢癌细胞发生凋亡。下调bcl-2的表达，增强bax的表达。结论诱导卵巢癌细胞发生凋亡是hTERT反义核酸抗卵巢癌作用的机制之一。hTERT反义核酸可能通过下调bcl-2的表达及增强bax的表达诱导卵巢癌细胞发生凋亡。     

反义技术在抗HBV感染中的应用

反义技术是近些年来随着现代分子生物学技术的发展而产生的新的生物医学治疗技术.它采用反义核酸分子抑制、封闭或破坏靶基因组的技术手段,包括反义寡核苷酸、核酶及RNA干扰等.反义分子通过与靶基因异性互补配对结合,阻断靶基因的复制、转录或翻译过程,从而发挥抗病毒作用.针对乙型肝炎病毒的反义技术也有了广泛而深入的研究.根据反义技术在分子、细胞以及动物水平上的研究表明:反义技术能够高效、特异地抑制HBV的复制与表达.     

反义核酸--研究基因功能的有效工具

反义核酸作为新药开发的重要途径 ,正被广泛应用于病毒性疾病、高血压病、心血管病、白血病等疾病治疗领域药物的研究中。目前 ,已有不少反义核酸药物进入临床 ,也已有经 FDA批准的新药上市。近年来 ,随着基因功能研究的深入和新基因的不断发现 ,作为基因表达抑制剂的反义核酸被越来越多地应用于与信号传导、细胞凋亡、胚胎发育等过程相关的基因功能的研究之中。迄今为止 ,已有针对诸如 ICAM- 1、p5 3、n NOS、cyclin D1、Rho A等多个基因分别应用反义核酸抑制其表达 ,进而确证基因功能的成功范例。随着反义技术的迅速完善 ,反义核酸必将在后基因组时代对基因功能的研究中发挥出更重要的作用。     

打点杂交及RNA酶保护分析法检测反义HSP90基因转染细胞中反义RNA的表达

目的：检测HSP90反义核酸转染细胞后反义RNA的表达。方法：采用打点杂交及RNA酶保护分析法。结果：HSP90反义核酸转染细胞AH－SGC7901,AH-SGC7901／VCR,AH－HCC7402及AH－Ec109有HSP90反义RNA的表达。结论：HSP90反义RNA在AH－SGC7901,AH－SGC7901／VCR,AH－HCC7402及AH－Ec109细胞的表达,为进一步研究HSP90在细胞生物学中的作用建立了可靠的细胞模型。     

反义治疗与白血病靶基因研究进展

反义技术是根据碱基互补原理 ,利用与目标靶ＤＮＡ或ＲＮＡ互补的短链片断封闭基因表达。反义技术用于抗白血病的理论基础 ,是用反义核酸中止原癌基因或癌基因的表达 ,使白血病细胞分化或凋亡 ,达到治疗的目的。其特点是 ,特异性强 ,对正常细胞无明显影响。反义核酸能在核酸水平上抗白血病 ,优于在蛋白水平的作用。基本内容包括 :反义ＲＮＡ、反义ＤＮＡ、核酶 (ｒｉｂｏｚｙｍｅ)和三股螺旋ＤＮＡ(ＤＮＡ -Ｔｒｉｐｌｅｘ)。主要通过下列作用之一或联合作用抑制靶基因表达 :1 与双链ＤＮＡ形成三链结构 ,使ＤＮＡ转录受阻。 2 结合靶ｍＲ…     

Antisense oligonucleotides in the study of central mechanisms of the cardiovascular regulation

In the past several years, the progress in the development of molecular biology and the elucidation of gene sequences has created new approaches for studying biological functions and developing new diagnostic and therapeutic strategies. One of the most exciting advances has been the development of antisense technology, which represents a new strategy allowing modulation of protein synthesis with high specificity by preventing protein expression at the level of RNA or DNA. Many classical pharmacological approaches in neurobiological research are often based on the inhibition of biologically active proteins, such as receptors for neurotransmitters, or enzymes involved in neurotransmitter synthesis or degradation. The use of antisense oligodeoxynucleotides offers an alternative tool to manipulate selectively the expression of neurotransmitters or their receptors in neuronal tissue. This approach is especially useful when selective, high-affinity antagonists are not available. As a result, this technology has gained acceptance in the study of cell signalling mechanisms and the molecular basis of neuronal function. This paper provides a brief background to the antisense technique and explores methodological aspects, particularly in the whole animal. The use of the antisense technology in studies focused on central mechanisms regulating the cardiovascular system is then discussed.     

基因沉默技术在斑马鱼研究中的应用

模式生物斑马鱼主要应用于探索遗传学、发育生物学和分子生物学等研究领域的分子机制,常用的研究策略是DNA诱变和基于RNA的基因沉默.但化学诱变技术和传统反义核酸技术存在缺陷,目前在应用中已受到很大限制.反义morpholino技术具有稳定、方便的特点,广泛应用于斑马鱼基因功能的研究.新兴的锌指核酶基因敲除技术不依赖于胚胎干细胞使其前景更为广阔.     

The use of synthetic polymers for delivery of therapeutic antisense oligodeoxynucleotides.

Developed over the past two decades, the antisense strategy has become a technology of recognised therapeutic potential, and many of the problems raised earlier in its application have been solved to varying extents. However, the adequate delivery of antisense oligodeoxynucleotides to individual cells remains an important and inordinately difficult challenge. Synthetic polymers appeared on this scene in the middle 1980s, and there is a surprisingly large variety used or proposed so far as agents for delivery of oligodeoxynucleotides. After discussing the principles of antisense strategy, certain aspects of the ingestion of macromolecules by cells, and the present situation of delivery procedures, this article analyses in detail the attempts to use synthetic polymers as carrier matrices and or cell membrane permeabilisation agents for delivery of antisense oligodeoxynucleotides. Structural aspects of various polymers, as well as the results, promises and limitations of their use are critically evaluated.     

Antisense Oligonucleotides in Cancer

The activation of dominant oncogenes and inactivation of tumour suppressor genes may result in cancer. These genetic events may represent novel targets for cancer therapy. Antisense nucleic acids can be used to modulate the expression of selected genes, and to suppress malignant behaviour in cancer cells. Nevertheless, in practice, the selection of suitable antisense targets still remains a trial-and-error procedure. Promising targets for antisense cancer therapy that have been extensively studied include proteases and protease receptors, telomerase, fusion genes, the Bcl family of proteins and various protein kinases. Combinations of antisense oligonucleotides with cytotoxic agents offer important advantages in cancer therapy. However, control oligonucleotides must be carefully chosen to separate the antisense effect from the many potential nonspecific effects. Several antisense drugs have been very effective in in vitro experiments, and have entered clinical trials. Successive generations of antisense drugs, including molecules with novel backbones or other structural modifications, chimeric oligonucleotides and peptide nucleic acids, are currently in development.     

Antisense studies of brain GABAA receptors

gamma-Aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain. The GABAA receptor complex, which is assumed to have a pentamer structure assembled from different polypeptide subunits, contains the binding sites for several clinically important compounds, e.g., the benzodiazepines and the barbiturates. A dysfunction of GABAergic inhibitory neurotransmission mediated via the GABAA receptor has been hypothesised to be a central factor in the pathogenesis of epilepsy. Antisense technology is based on the possibility of selectively inhibiting gene expression at the level of messengerRNA (mRNA). An antisense oligodeoxy nucleotide (ODN), a short synthetic single-stranded DNA molecule, is believed to inhibit the biosynthesis of a particular protein via nucleotide specific hybridisation to the mRNA encoding the protein. Antisense ODNs are used as tools for the investigation of the physiological roles played by individual proteins. The aim of the present study was to investigate the feasibility of selectively inhibiting the expression of a major subunit of the GABAA receptor complex in the rat brain in vivo by means of antisense technology. The thesis describes the changes observed following intrahippocampal administration of antisense ODN targeted to the GABAA receptor gamma 2 subunit. This subunit is a constituent of the majority of GABAA receptor complexes in the brain. Biochemical, morphological, electroencephalographic and behavioural changes induced by the antisense ODN treatment are described. The results support the notion that the primary event induced by the antisense ODN is a specific down-regulation of the gamma 2 subunit protein and that this leads to a decrease in the number of functional GABAA receptors and a state of diminished hippocampal GABAergic inhibitory neurotransmission. Antisense ODN-treated rats spontaneously develop limbic status epilepticus; prolonged antisense ODN treatment results in severe neurodegenerative changes in the hippocampus. The results of the study support the hypothesis that the GABAA receptor is critically involved in epileptogenesis. The results are viewed as a contribution to the understanding of the GABAA receptor complex and of mechanisms of epileptic phenomena and neuronal cell death. The presented animal model is suggested as a pathophysiologically relevant model of temporal lobe epilepsy and limbic status epilepticus. The results may also be of value for the general characterisation of antisense technology as a neuroscientific tool.     

Insulin-like growth factor type I biology and targeting in malignant gliomas

Growth factors such as insulin-like growth factor type I (IGF-I), epidermal growth factor (EGF), vascular-endothelial growth factor (VEGF) and transforming growth factor beta (TGF-beta) are present during the development of the CNS. When they reappear in the mature brain they are overexpressed in neoplastic glia, participating in the development of the most common human brain malignant tumor, glioblastoma multiforme, which is invariably fatal. Progress in treatment of this disease involves an increase in median survival from 8 to 11 months to an average of 15 months, rarely to 18 months. We do not know any therapy, which can make a complete stop of this neoplasm. To inhibit this process various anti-growth factor therapies have been proposed. We describe actual applications of growth factor inhibitors and antisense approaches. The review highlights results obtained with the promising treatment of glioblastoma multiforme: using inhibitors and antisense targeting growth factors, including IGF-I, their receptors, and their downstream signaling effectors including glycogenesis and oncogenes. The antisense strategies have been the subject of many clinical trials, especially the IGF-I antisense approach. Such antisense therapies, already introduced in clinical trial in the USA, Europe and Asia, will soon become the preferred alternative treatment for human glioblastoma multiforme. The inhibition of signal transduction pathways common to growth factors and glycogenesis appears as a parallel challenge to glioblastoma multiforme inhibition studies.     

Inhibition of Plasmodium falciparum clag9 gene function by antisense RNA

We have previously shown by targeted gene disruption that the clag9 gene of Plasmodium falciparum is essential for cytoadherence to CD36. Here we report inhibition of the function of clag9 by the use of an antisense RNA vector as an alternative to targeted gene disruption. We transfected an antisense construct of clag9 into the P. falciparum clone 3D7 and when the resulting line was cultured in the presence of pyrimethamine it showed 15-fold lower cytoadherence to C32 melanoma cells than the control. Reversion to wildtype upon removal of the introduced plasmid provides direct evidence that the event responsible for the phenotypic change is not at an unrelated site and this approach provides a valuable new tool in malaria transfection technology.     

Targeted delivery of antisense oligonucleotides by molecular conjugates

Antisense oligonucleotides efficiently inhibit gene expression in vitro; however, the successful therapeutic application of this technology in vivo will require the development of improved delivery systems. In this report we describe a technique that efficiently delivers antisense oligonucleotides into cells using molecular conjugates. This technique, which was initially developed for the delivery of eukaryotic genes, is based on the construction of DNA-protein complexes that are recognized by the liver-specific asialoglycoprotein receptor. Binding of poly(l-lysine)-asialoorosomucoid (AsOR) protein conjugates with phosphorothioate antisense oligonucleotides to chloramphenicol acetyltransferase (CAT) led to the formation of 50- to 150-nm toroids. Exposure of the antisense molecular complexes (3 µM oligonucleotide) to NIH 3T3 cells genetically modified to express both the AsOR receptor and CAT, inhibited CAT expression by 54%, which was completely blocked by excess AsOR. Equivalent inhibition of CAT activity with purified oligonucleotide alone was observed at a 30 µM concentration. Furthermore, examination of the cells using indirect immunofluorescence for the presence of CAT protein showed 28% of cells exposed to the molecular conjugates lacked any detectable CAT enzyme. Cells exposed to oligonucleotide alone showed a highly variable staining pattern, and only a few of the cells were completely void of CAT protein. Together these data demonstrate that molecular conjugates provide a highly specific and efficient system for the delivery of antisense oligonucleotides.     

Implication of bax in Xenopus laevis tail regression at metamorphosis.

Apoptosis is fundamental to normal vertebrate development. A dramatic example of postembryonic development involving apoptosis is tail regression during amphibian metamorphosis. Earlier studies led us to propose a functional role for the pro-apoptotic protein Bax in tadpole tail regression. However, its physiological relevance has never been analyzed. We have now cloned a cDNA encoding Xenopus laevis bax (xlbax) and used in vivo gene transfer in tail muscle to analyze the effects of xlbax overexpression. Furthermore, by using an antisense strategy in a similar experimental paradigm, xlbax antisense mRNA was shown to block the apoptotic effects of xlbax and protect against apoptosis in metamorphosing tadpoles. Our results suggest that xlbax is a regulator of muscle fiber death in the regressing tail during metamorphosis.     

Antisense oligonucleotides and prevention of tumor growth: a different approach and proposal for a new method

There have been several attempts to prevent tumor formation and growth. However, none of the developed methods gives a completely satisfying result for the treatment of tumor masses. The most often used therapies against tumor cells are radiotherapy and chemotherapy. However, utilization of these methods to treat cancer generally result in generation of undesired side effects. In recent years, the antisense oligonucleotide technology has been employed, with success to an extent, in prevention of tumor growth. However, this method has its limitations. One of the most important limitation is that all of the crucial genes that play certain roles and are specifically expressed in tumor cells have not yet been identified. Therefore, only a few numbers of genes that are shown to play a role in tumor cells are targeted by the antisense oligonucleotide method. The aim of the present study is to propose a hypotheses and outline the involved procedure which could be used to generate oligonucleotides that are antisense to genes or mRNAs that display certain specific functions in tumor cells but are yet to be identified. The proposed hypotheses involves first, a careful isolation of differentially expressed mRNAs by using the tumor and the corresponding normal cells. These mRNAs should represent the genes that operate in tumor cells but not in the corresponding normal cells. Following the isolation of the differentially expressed mRNAs, they will be reverse transcribed and the desired amounts of cDNA copies will be obtained. The cDNA copies will then be used differentially as a source for oligonucleotides that are antisense to genes or mRNAs. To obtain the desired length oligonucleotides that will be used as antisense oligonucleotides the cDNA copies will be subjected to Maxam-Gilbert fragmentation and/or controlled enodonuclease digestion. These two mentioned procedures could be optimized and used together or separately to obtain the desired length oligonucleotides that will be used against tumor cells.