Irritable bowel syndrome is a gastrointestinal disorder of unknown etiology characterized by widespread, chronic abdominal pain associated with altered bowel movements. Increasing amounts of evidence indicate that injury and inflammation during the neonatal period have long-term effects on tissue structure and function in the adult that may predispose to gastrointestinal diseases. In this study we aimed to investigate how the epigenetic regulation of DNA demethylation of the p2x7r locus guided by the transcription factor GATA binding protein 1 (GATA1) in spinal astrocytes affects chronic visceral pain in adult rats with neonatal colonic inflammation (NCI). The spinal GATA1 targeting to DNA demethylation of p2x7r locus in these rats was assessed by assessing GATA1 function with luciferase assay, chromatin immunoprecipitation, patch clamp, and interference in vitro and in vivo. In addition, a decoy oligodeoxynucleotide was designed and applied to determine the influence of GATA1 on the DNA methylation of a p2x7r CpG island. We showed that NCI caused the induction of GATA1, Ten-eleven translocation 3 (TET3), and purinergic receptors (P2X7Rs) in astrocytes of the spinal dorsal horn, and demonstrated that inhibiting these molecules markedly increased the pain threshold, inhibited the activation of astrocytes, and decreased the spinal sEPSC frequency. NCI also markedly demethylated the p2x7r locus in a manner dependent on the enhancement of both a GATA1-TET3 physical interaction and GATA1 binding at the p2x7r promoter. Importantly, we showed that demethylation of the p2x7r locus (and the attendant increase in P2X7R expression) was reversed upon knockdown of GATA1 or TET3 expression, and demonstrated that a decoy oligodeoxynucleotide that selectively blocked the GATA1 binding site increased the methylation of a CpG island in the p2x7r promoter. These results demonstrate that chronic visceral pain is mediated synergistically by GATA1 and TET3 via a DNA-demethylation mechanism that controls p2x7r transcription in spinal dorsal horn astrocytes, and provide a potential therapeutic strategy by targeting GATA1 and p2x7r locus binding.
Animals ; Astrocytes/metabolism* ; DNA Demethylation ; Epigenesis, Genetic ; GATA1 Transcription Factor/metabolism* ; Inflammation/metabolism* ; Oligodeoxyribonucleotides/metabolism* ; Rats ; Rats, Sprague-Dawley ; Receptors, Purinergic P2X7/metabolism* ; Visceral Pain/metabolism*

Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.
Antiviral Agents ; pharmacology ; Aptamers, Nucleotide ; pharmacology ; therapeutic use ; Genome, Viral ; drug effects ; HIV ; drug effects ; HIV Reverse Transcriptase ; metabolism ; Hepacivirus ; drug effects ; genetics ; Humans ; Macular Degeneration ; drug therapy ; Neoplasms ; drug therapy ; Oligodeoxyribonucleotides ; therapeutic use ; RNA, Small Interfering ; pharmacology ; SELEX Aptamer Technique ; Viral Envelope Proteins ; metabolism ; Virus Replication ; drug effects

OBJECTIVETo investigate the protective effect of suppressive oligodeoxynucleotides (Sup ODN) on interferon-γ (IFN-γ) and signal transducers and activators of transcription (pSTAT4) expression of Silica-induced pulmonary inflammation in Mice.

METHODSSixty Balb/c mice were randomly divided into 4 groups, normal control group, silicious group, suppressive oligodeoxynucleotides (Sup ODN) group, control oligodeoxynucleotides (Con ODN) group. Except the normal control group injected normal saline, the rest groups were induced by the intratracheal instillation of 0.1 ml (5 g/L) of sterilized silica suspension. Sup ODN group and Con ODN group were treated by i.p. injection of 0.3 ml (1mg/mL) of suppressive or control ODN 3 h before silica administration. After 7 days, the animals were killed and levels of IFN-γ were detected by ELISA. The pathologic changes in lung tissues of mice were observed with HE staining. Expressions of IFN-γ and pSTAT4 in lung tissue were detected with immunohistochemistry and quantified by Image-Pro Plus 7.0.

RESULTSHE staining showed that the lung tissue of silicious group were damaged seriously than Sup ODN group. Compared with the normal control group (serum: (280.1±41.3) pg/ml, lung tissue: (0.249±0.373), IFN-γ increased in silicious group (serum: (886.3±81.7) pg/ml, lung tissue: (0.270±0.300) (P < 0.05). Compared with the normal control group and Con ODN group [(894.5±91.6) pg/ml], IFN-γ in the serum of Sup ODN group decreased significantly (P < 0.01). Compared with the silicious group , IFN-γ in lung tissue decreased in Sup ODN group (0.241±0.250) (P < 0.05). Compared with the normal control group (0.279±0.353), pSTAT4 in lung tissue increased significantly in silicious group (0.313±0.231) (P < 0.01). Compared with the silicious group, pSTAT4 in lung tissue decreased significantly in Sup ODN group (0.269±0.523) (P < 0.01).

CONCLUSIONSup ODN attained protective effect on Silica treated mice by suppressing expression of IFN-γ and pSTAT4.

Animals ; Female ; Inflammation ; metabolism ; Interferon-gamma ; metabolism ; Lung ; drug effects ; metabolism ; pathology ; Mice ; Mice, Inbred BALB C ; Oligodeoxyribonucleotides ; pharmacology ; Phosphorylation ; STAT4 Transcription Factor ; metabolism ; Silicon Dioxide ; toxicity

OBJECTIVETo investigate the high expression of HPV16L2N120E7E6 fusion protein by prokaryotic expression system, and evaluate its immunogenicity and antitumor efficacy in vaccinated mice.

METHODSThe HPV16L2N120E7E6 fusion gene, its codons were optimized to increase the expression of the protein, was constructed by overlap extension PCR and inserted into prokaryotic expression vector pET9a. Then the fusion protein was expressed by inducing with IPTG in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and further detected by SDS-PAGE and Western-blot. Finally, the humoral and cellular immune responses were measured by ELISA and ELISPOT, respectively, in vaccinated mice with the purified HPV16L2N120E7E6 fusion protein, and the antitumor efficacy was assessed in mice using the TC-1 tumor challenge model.

RESULTSThe codon-optimized HPV16L2N120E7E6 fusion gene was highly expressed in E. coli strain BL21 (DE3) harboring with plasmid pETL2N120E7E6, and the amount of fusion protein was nearly 48.6% of the total bacterial protein. The purified fusion protein could induce high titer of specific antibody against L2, E7 and E6 in vaccinated mice. When accompanied with the adjuvant CpG, the fusion protein was able to elicit strong and moderate cellular immune responses in vaccinated mice against peptide HPV16E7(49-57) and peptide pools of HPV16E6, respectively. Furthermore, the tumor therapeutic experiment showed that HPV16L2N120E7E6 + CpG could prevent the tumor formation in 80.0% (8/10) vaccinated mice.

CONCLUSIONSThe data of this study suggest that HPV16L2N120E7E6 fusion protein could be a promising candidate vaccine for treatment of chronic HPV16 infection and post-operative adjuvant therapy for cervical cancer.

Adjuvants, Immunologic ; pharmacology ; Animals ; Cancer Vaccines ; immunology ; therapeutic use ; Capsid Proteins ; genetics ; immunology ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Codon ; Escherichia coli ; immunology ; metabolism ; Female ; Humans ; Immunization ; methods ; Immunotherapy ; methods ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Oligodeoxyribonucleotides ; immunology ; Oncogene Proteins, Viral ; genetics ; immunology ; metabolism ; Papillomavirus E7 Proteins ; genetics ; immunology ; metabolism ; Papillomavirus Vaccines ; immunology ; therapeutic use ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Repressor Proteins ; genetics ; immunology ; metabolism

OBJECTIVE: To investigate the changes of mitochondrion by transferring antisense oligodeoxynucleotide Stat3 into laryngeal carcinoma Hep-2 cell, for elucidating the mechanism of laryngeal carcinoma Hep-2 cell apoptosis, for developing more effective treatment for laryngeal cancer. METHOD: The designed Stat3 ASODN was transferred into laryngeal carcinoma Hep-2 cell by lipofection. Mitochondrion membrane potential, VDAC and Cyt-c were detected for determining the changes of mitochondrion. RESULT: MMP was fell, Cyt-c and VDAC were increased with the heighten concentration of ASODN. CONCLUSION Mitochondria approach play an important role in the apoptosis mechanism of human Hep-2 cell by Stat3. This research elucidated the regulating mechanism of Hep-2 cell proliferation by Stat3, provided a new research focus for clinical therapy.
Apoptosis ; Cell Proliferation ; Cytochromes c ; metabolism ; Hep G2 Cells ; Humans ; Laryngeal Neoplasms ; genetics ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism ; Oligodeoxyribonucleotides, Antisense ; genetics ; STAT3 Transcription Factor ; genetics ; Transfection ; Voltage-Dependent Anion Channels ; metabolism

CD8-Positive T-Lymphocytes ; metabolism ; Case-Control Studies ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Interferon-gamma ; biosynthesis ; Lymphocytes ; immunology ; Oligodeoxyribonucleotides ; pharmacology

OBJECTIVETo explore the effects of antisense oligodeoxynucleotides (ASODN) on proliferation and apoptosis in gastric cancer cell line BGC-823 cells and the molecular mechanisms induced by ASODN.

METHODSsurvivin ASODN-1, survivin ASODN-2 and survivin ASODN-3 were transfected into BGC-823 cells by Lipofectamine(TM) 2000 transfection reagent. The growth activity of BGC-823 cells was detected by MTT assay. Apoptosis index (AI), proliferation index (PI), cell cycle and expressions of survivin, VEGF and Smac/DIABLO proteins were detected by flow cytometry (FCM). The changes of survivin mRNA, VEGF mRNA and Smac/DIABLO mRNA were detected by RT-PCR.

RESULTSThe expression of survivin was down-regulated by the three ASODN sequences, especially the ASODN-2 was best. At 48 hours after transfection with 600 nmol/L survivin ASODN-2, the cells in G(1)/G(0) phase were significantly increased [(72.25 ± 2.95)%], apoptotic index increased [(11.31 ± 0.38)%], proliferation index decreased [(27.77 ± 2.97)%], compared with those in the control group [(56.25 ± 0.75)%, (1.62 ± 0.36)%, (43.80 ± 0.80)%, all P < 0.05]. The survivin mRNA and protein levels (0.523 ± 0.091, 0.733 ± 0.009) were down-regulated compared with those in the control group (0.861 ± 0.047, 0.997 ± 0.233), VEGF (0.519 ± 0.076, 0.75 ± 0.006) were down-regulated compared with those in the control group (0.779 ± 0.059, 1.000 ± 0.01), while those of Smac/DIABLO(0.899 ± 0.113, 1.637 ± 0.023)were up-regulated compared with those in the control group (0.558 ± 0.041, 1.000 ± 0.049, all P < 0.05).

CONCLUSIONSSurvivin ASODN can induce apoptosis and inhibit the proliferation of gastric cancer cell line BGC-823 cells. Those effects are induced through up-regulation of Smac/DIABLO and down-regulation of survivin and VEGF expression simultaneously.

Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Humans ; Inhibitor of Apoptosis Proteins ; genetics ; metabolism ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Mitochondrial Proteins ; genetics ; metabolism ; Oligodeoxyribonucleotides, Antisense ; genetics ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Transfection ; Up-Regulation ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.
Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; DNA, Antisense ; chemistry ; genetics ; Female ; Ferric Compounds ; chemistry ; Genes, erbB-2 ; genetics ; Humans ; Magnetics ; Microscopy, Atomic Force ; methods ; Molecular Probe Techniques ; Nucleic Acid Probes ; chemistry ; genetics ; Oligodeoxyribonucleotides ; chemistry ; genetics ; Oxyphil Cells ; ultrastructure ; RNA, Messenger ; genetics ; metabolism

We have investigated the effect of various forms of phosphodiester cytidine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs) on the production of pro-inflammatory cytokines and related genes in RAW 264.7 macrophages. Treatment with the CpG ODNs increased the expression of tumor necrosis factor alpha (TNF-alpha), IL-6, and inducible nitric oxide synthase but not interleukin-1beta (IL-1beta). We also investigated the effect of CpG ODNs on the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) genes which are known to facilitate cholesterol efflux from macrophages for anti-atherosclerosis. CpG 2006 significantly reduced the levels of ABCG1 mRNA as determined by real-time polymerase chain reaction, whereas ABCA1 mRNA level was not changed. Western blot analysis further confirmed the reduction of ABCG1 protein expression by CpG 2006. In addition, we also determined the protein level of peroxisome proliferator activated receptor gamma (PPARgamma), which is recognized as a transcriptional activator of ABC transporters, was also reduced by CpG 2006. Thus, these results suggest that ABCG1 is specifically down-regulated by CpG 2006 in a PPARgamma-dependent manner in macrophages.
ATP-Binding Cassette Transporters/drug effects/genetics/*metabolism ; Animals ; Atherosclerosis/metabolism ; Cholesterol/metabolism ; Cytokines/drug effects/metabolism ; Gene Expression Regulation ; Inflammation/*metabolism ; Interleukin-1beta/drug effects/metabolism ; Interleukin-6/metabolism ; Lipoproteins/drug effects/genetics/*metabolism ; Macrophages/*cytology/*metabolism ; Mice ; Nitric Oxide Synthase/drug effects/metabolism ; Oligodeoxyribonucleotides/*pharmacology ; PPAR gamma/genetics/*metabolism ; Tumor Necrosis Factor-alpha/drug effects/metabolism

Secondary lymphoid tissue chemokine (SLC), which is expressed in T cell zones of secondary lymphoid organs, including the spleen and lymph nodes, strongly recruits both T lymphocytes and mature dendritic cells. As appropriate interaction of tumor-specific T cells and mature dendritic cells, equipped with tumor antigens, is a prerequisite for effective T cell immunity against established tumors, we mobilized lymphocytes and dendritic cells to tumor sites by intratumoral injection of secondary lymphoid tissue chemokine-Fc (SLC-Fc) fusion protein using the B16F10 murine melanoma model. Activation of dendritic cells, another prerequisite for the effective activation of naive tumor-specific T cells, was achieved by the addition of immunostimulatory cytosine-phosphorothioate-guanine oligodeoxynucleotide (CpG-ODN) into the tumor site. Intratumoral administration of SLC-Fc or CpG-ODN revealed antitumor effects against B16F10 murine melanoma grown in the subcutaneous space. Co-treatment of SLC-Fc and CpG-ODN displayed synergistic effects in reducing the tumor size. The synergistic antitumor effect in co-treatment group was correlated with the synergistic/additive increase in the infiltration of CD4+ T cells and CD11c+ dendritic cells in the tumor mass compared to the single treatment groups. These results suggest that the combined use of chemokines and adjuvant molecules may be a possible strategy in clinical tumor immunotherapy.
Animals ; Antigens, CD11c/immunology ; CD4-Positive T-Lymphocytes/immunology ; Cell Line, Tumor ; Cell Proliferation/drug effects ; Chemokine CCL21/*administration & dosage/pharmacology ; Chemotaxis, Leukocyte ; Dendritic Cells/immunology/metabolism ; Immunotherapy ; Injections, Intralesional ; Melanoma, Experimental/*immunology/*therapy ; Mice ; Mice, Inbred C57BL ; Oligodeoxyribonucleotides/*administration & dosage/pharmacology ; T-Lymphocytes/immunology/metabolism