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Journal of Medical Colleges of PLA(China) 1997年第3期12卷 243-248页

作者：丁天兵马文煜杨涌峰张明杰

To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences of the first AUG codon and 5’ terminus of NSS of JEV SA--14 genome have been synthesized and their effects on CPE, viral antigen expression and virus plaque formation were tested in vitro. Results: The resultsshowed that 1. 0 pmol/L of S-ODN greatly deferred the onset of CPE in cultured BHKZI cells for at least 48 h.Addition of 5. 0 pmol/L or more S--ODN to culture medium fluid, 2 h prior to 100TCID,,virus inoculum, notablysuppressed viral antigen expression in the cells by making it lower than the limit of EIA detection in 48 h. The inhibition lasted more than 96 h. Viral plaque assay results demonstrated that S-ODN were most effect’ive within 18h with plaque inhibition rate over 90% by 5. 0 pmol/L S--ODNI. The inhibitory activity soon faded in 24 h. In addition, high concentrations (up to 80. 0 pmol/L) of S--ODN did not show any obvious cytotoxicity in 6 d by usingtrypan blue dye exclusion method. Conclusion: The specific synthetic S--ODN transitorily inhibited JEV replicationin BHK--ZI cells with characteristics of specificity and S--ODN dose--dependence.

关键词： Japanese encephalitis virus oligodeoxynucleotide, antisense antiviral agent

Following Inhibition of BCL-2 by antisense Oligonucleotides Compensatory Suppression of Apoptosis Involves the Direct Signal Transduction Pathway of LNCaP Cells

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Open Journal of Apoptosis 2015年第1期4卷 1-10页

作者： Marvin Rubenstein Courtney M. P. Hollowell Patrick Guinan Division of Cellular Biology Hektoen Institute for Medical Research Chicago IL USA Department of Biochemistry Rush University Medical Center Chicago IL USA Department of Urology Rush University Medical Center Chicago IL USA Division of Urology Stroger Hospital of Cook County Chicago IL USA Department of Urology University of Illinois at Chicago Chicago IL USA

Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and promote tumor aggressiveness. In addition to the inhibition of BCL-2, we find that the apoptosis promoter caspase-3 activity is suppressed, the transcription activity of STAT-3 is enhanced, while other regulators (bax, clusterin, AKT-1) associated with mitochondrial regulated apoptosis and caspase cascade are either unchanged or undetectable. We now evaluate proteins associated with the second pathway of apoptosis activation mediated by direct signal transduction involving fas, fas-ligand (a tumor necrosis factor-like cell surface receptor aka CD95), as well as the similar programmed death cell surface receptor (PD-1) and its respective ligand (PD-1L). This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)];and employing RT-PCR. The expression of these four proteins was evaluated. Expression of fas-ligand, PD-1 and PD-L1 were all significantly enhanced, whereas fas itself was undetectable. This suggests that in addition to pathways associated with the mitochondrial pathway of apoptosis, compensatory changes occur in the direct signal transduction pathway of this process. In addition to alterations in androgen sensitivity, growth factor expression and oncogene expression, these data suggest that suppressive BCL-2 therapy involves multiple pathways, including those involved with immune targeting and cytotoxicity and must be taken into account to make gene therapy more efficacious.

关键词： antisense Oligonucleotides Prostate Cancer Fas Fas-Ligand PD-1 PD-1 Ligand Caspase-3 BCL-2 Bax Therapy

HSP90β participates in regulation of heat shock gene expression

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Chinese Science Bulletin 1996年第20期41卷 1730-1734页

作者：王艳林沈珝琲梅树恩刘巨洪张书岭吴宁华 State Key Laboratory of Medical Molecular Biology Chinese Academy of Medical Sciences Peking Union Medical College Beijing 100005 China

Heat shock proteins (HSP) are a group of proteins highly conserved in evolution.They play important physiological roles, usually as molecular chaperones in living cells, and take great parts in cell protection process against stress, and in signal transduction as well.HSP70 and HSP90 are the major HSPs expressed in human peripheral lymphocytes upon heat shock. HSP90 consists of two copies, designated as HSP90α and HSP90β. Heat shock factors (HSF), the trans regulators, participate in the regulation of HSP gene transcription. Different from HSP70 which is dramatically induced by stress, both heat shock and cold shock, HSP90 expresses at a relatively high level physiologically and may exist as complexes with various steroid receptors or some important components in signal transduction pathway, and plays a role of specific molecular chaperone. The aim of

关键词： antisense cDNA hsp90 gene molecular chaperone.

Lignin biosynthesis by suppression of two O-methyl-transferases

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Chinese Science Bulletin 2002年第13期47卷 1092-1095,1145页

作者： ZHAO Huayan WEI Jianhua ZHANG Jinyu LIU Huirong WANG Tai SONG Yanru Institute of Botany Chinese Academy of SciencesBeijing 100093China Beijing Center of Agro-Biotechnology Beijing Institute of Agriculture and ForestryBeijing 100089China 不详

Caffeic acid O-methyltransferase (COMT) and caffeoyl-CoA-3-O-methyltransferase (CCoAOMT) genes encode two methyltransferases at different substrate levels in lignin biosynthesis. We constructed two single antisense expression vectors containing the cDNA of COMT or CCoAOMT genes and one dual antisense expression vector containing cDNAs of both OMTs genes. The antisense constructs were transferred into tobacco mediated by Agrobac-terium tumefacience. PCR-Southern analysis indicated that antisense cDNAs had been integrated into the genome of the transgenic tobacco. The antisense genes were also expressed at transcriptional level displayed by Northern dot analysis. Klason lignin assay of 3-month-old transgenic tobaccos showed that repression in COMT or CCoAOMT alone could result in reduction of lignin content, more reduction was caused by suppression in CCoAOMT than in COMT. Furthermore, simultaneous suppression of both COMT and CCoAOMT resulted in more reduction than that of single gene, which indicated

关键词： COMT CCoAOMT antisense RNA lignin histochemi-cal staining.

Generation of bcl-2 antisense RNA promotes apoptosis of human promyelocytic leukemia cell line HL-60

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Journal of Medical Colleges of PLA(China) 1997年第2期12卷 110-115页

作者：朱峰王成济惠宏襄赵永同金明

Objective: To analyze the effect of bcl-2 antisense RNA on the apoptosis of promyelocytic cell line HL-60. Methods: A plasmid pDOR-AB containing bcl-2 antisense cDNA was trans fected into HL-6O cells by lipofectin, and the effect of transfection was assured by DNA and RNA dot blottingI the change of bcl-2 expression and cell cycle was tested by flow cytometry; a morphologica1 change was observed by light microscope and electron microscope; and finally the sensitivity of trans fected cells to etoposide was compared with that of non-trans fected cells by gel electrophoresis. Results: pDOR-AB was successfully trans fected into HL-6o cells and its transcript was observed; Bcl-2 was down-regulated significantly ; apoptosis peak appeared before G1 phase in flow cy-tometry analysis: apoptotic cells could be seen by electron microscope, and during DNA gel electrophoresis the DNA ladder apppeared more frequently in the group trans fected with pDOR-AB than in transfected with pDOR and untransfected groups. Conclusion: Transient expression of bcl-2 antisense RNA can promote apoptosis of HL-60 cells and bco-2 plays a key role in the apoptosis of HL-60 cells.

关键词： bcl-21 antisense RNA HL-60 cells apoptosis

Murine neurofibroma reversion by antisense RNA for HTLV-I tax

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Science China(Life Sciences) 1999年第1期42卷 8-16页

作者：李昌本 Mark C.Horowitz Nancy H.Ruddle Department of Orthopaedics and Rehabilitation Yale University Schcol of Medicine New Haven Connecticut 06520 USA Department of Epidemiology and Public Health Yale University School of Medicine New Haven Connecticut 06520 USA

Neurofibroma cell lines derived from mice transgenic for HTLV-I LTR tax express high levels of HTLV-I tax mRNA and protein and exhibit a transformed phenotype. A retrovirus vector carrying HTLV-I tax cDNA in reversed transcriptional orientation was stably transfected into the neurofibroma cells. antisense RNA inhibited expression of the tax gene with a decrease of more than 40 % in both tax mRNA and protein. Tax antisense RNA reversed the transformed phenotype as exhibited by dramatic changes in cell morphology and growth characteristics. Expression of several cellular genes which are activated by Tax protein including GM-CSF, IL-6, LT/TNF, c-myc and LIF was down-regulated, while M-CSF and c-src proto-oncogene expressions were up-regulated. Accumulation of β-actin mRNA was not affected. The changes that occurred in the tax antisense expressing neurofibroma cells could be the consequence of the decreased concentration of Tax protein. These results also indicate that HTLV-I Tax protein is crucial for

关键词： neurofibroma antisense RNA HTLV-I tax cytokine proto-oncogene.

Construction of antisense RNA expression vectors and correction of splicing defect in human β-globin gene (IVS-2-654 C→T mutant) in HeLa cells

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Science China(Life Sciences) 1998年第1期41卷 99-106页

作者：宫澜顾小锋王敏黄淑帧曾溢滔 Shanghai Institute of Medical Genetics Shanghai Children''s Hospital China

The antisense fragments, which were available in in vitro system, were cloned into the mammalian expression vector pcDNA3, and were transfected into H654 cells, a mammalian cell line stably expressing the thalassaemic (IVS 2 654 C→T) human β globin gene. In these transfected cells, the level of correctly spliced β globin mRNA in total β globin mRNA (β/(β+β *)) was improved from 0.07 (0 d) to 0.22 (3 d), and this effect persisted for up to 15 d post transfection. All the results demonstrated that antisense RNAs were able to be transcribed from the antisense fragment expression vectors stably and effectively suppressed aberrant splicing pattern of the mutated β globin gene (IVS 2 654 C→T) and restored correct splicing pathway. This work provided a novel approach with potential clinical significance to gene therapy of this kind of splicing mutants including β thalassaemia (IVS 2 654 C→T) by antisense RNAs.

关键词： antisense RNAs thalassemia gene therapy splicing defect.

Immunotherapy of Malignant Tumors Using antisense Anti-IGF-I Approach: Case of Glioblastoma

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Journal of Cancer Therapy 2014年第7期5卷 685-705页

作者： Annabelle Trojan Lina M. Jay Heliodor Kasprzak Donald D. Anthony Jerzy Trojan Department of General Medical Sciences Case Western Reserve University Cleveland USA Department of Investigation National Institute of Health—INS Bogota Colombia Department of Neurosurgery CM Nicolas Copernic University Bydgoszcz Poland Faculty of Medicine CURN University Cartagena de Indias Colombia

The review article describes the criteria established for methodology of antisense anti IGF-I therapy of malignant tumors, particularly of glioblastoma. The cancer patients, after classical therapy of surgery, radiotherapy and chemotherapy, have undergone the injection of genetically modified autologous malignant cells—transfected by IGF-I antisense/triple helix expression vectors. For all cancer patients supervised for up to 19 months, the period corresponding to minimum survival of glioblastoma patients, the following common immune criteria for “anti IGF-I” strategy were admitted: 1) characteristics of cell “vaccines”—absence of IGF-I and expression of MHC-I in cloned transfected cells;2) the peripheral blood lymphocytes, PBL cells, removed after every of two successive vaccinations, demonstrate an increasing level of CD8+ and CD8+28+ molecules (with a switch from CD8+11b+ to CD8+11b-).

关键词： Immunogene Therapy Malignant Tumors Glioblastoma IGF-I antisense

Genetic Transformation of <i>Citrus sinensis</i>L. with an <i>antisense ACC oxidase</i>Gene

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American Journal of Plant Sciences 2012年第9期3卷 1336-1340页

作者： Sumontip Bunnag Duangkamol Tangpong Department of Biology Faculty of Science Khon Kaen University Khon Kaen Thailand

This work was carried out to optimize the conditions for highly effective embryogenic callus induction from mature seeds, plantlet regeneration and genetic transformation of Citrus sinensis L. by Agrobacterium tumefaciens strain EHA105 (pCAMBIA 1305.1). Embryogenic calli could be successfully induced from mature seeds employing the MT medium supplemented with 500 mg/l malt extract. The percentage of embryogenic callus induction was 85. With the same medium, the high proliferation rate of embryogenic callus was achieved. The liquid MT medium containing 500 mg/l malt extract in combination with 50 mg/l lactose could be used as the embryoid development medium. Somatic embryos, however, could be regenerated with normal shoots and roots in the MS medium, with the regeneration percentage of 60. The delivery of an antisense ACC oxidase gene into the species C. sinensis mediated by Agrobacterium tumefaciens strain EHA 105 was successful by co-cultivating explants with the strain EHA 105 for 10 min, following that by eliminating the bacterium with 200 mg/l cefotaxime, and subsequently selecting transformed embryoid with 20 mg/l hygromycin. Verified histochemically by GUS assay, putative transformants showed the percentage of gus gene expression of 100. Molecular analysis using PCR confirmed the integration of the antisense ACC oxidase gene into plant genome.

关键词： Citrus sinensis antisense ACC oxidase Agrobacterium tumefaciens Cefotaxime

Inhibition of α_1(Ⅰ) collagen gene in vitro transcription by antisense oligodeoxynucleotides

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Journal of Medical Colleges of PLA(China) 2000年第3期15卷 176-177,181页

作者：单越新罗超权徐钤利天增

Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.

关键词： α1(Ⅰ) collagen gene antisense oligodeoxynucleotides in vitro transcription