OBJECTIVETo investigate the interference and the mechanisms of Bcl-2 and HER-2 genes antisense oligodeoxynucleotides combined transfection in the human tongue carcinoma Tca8113 cell lines.

METHODSThere were 6 groups in our study, normal control group, Bcl-2 sense experimental group, HER-2 sense experimental group, Bcl-2 antisense experimental group, HER-2 antisense experimental group, Bcl-2 and HER-2 genes antisense oligodeoxynucleotides combined transfection experimental group. In the different times after liposome-mediated transfection, the cell apoptosis, Bcl-2 and HER-2 expressing level were observed by RT-PCR and electronic microscope.

RESULTSAccording to the results of combined transfection experimental group, the apoptosis body and apoptosis cells were observed. The expression of genes were decreased statistically in both Bcl-2 and HER-2, respectively. Bcl-2 and HER-2 combined ASODN was superior to single ASODN in the intervention of tongue carcinoma.

CONCLUSIONBcl-2 and HER-2 ASODN can effectively interfere the expression of HER-2 and Bcl-2 genes. The expression of HER-2 and Bcl-2 can be reduced, and the apoptosis of cells can be enhanced.

Apoptosis ; Female ; Genes, erbB-2 ; Humans ; Oligodeoxyribonucleotides ; Oligodeoxyribonucleotides, Antisense ; Transfection

AIMTo investigate the effect of inhibition of telomerase with human telomerase reverse transcriptase (hTERT) antisense on tumor necrosis factor-alpha (TNF-alpha)-induced apoptosis in prostate cancer cells (PC3).

METHODSAntisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and purified. Telomerase activity was measured using the telomeric repeat amplification protocol (TRAP) and polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). hTERT mRNA was measured by reverse transcription PCR (RT-PCR) assay and gel-image system. hTERT protein was detected by immunochemistry and flow cytometry. Cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium (MTT) assay. Cell apoptosis was observed by morphological method and determined by flow cytometry.

RESULTSThe telomerase activity decreased with time after hTERT AS PS-ODN treatment. The levels of hTERT mRNA decreased with time after hTERT AS PS-ODN treatment, which appeared before the decline of the telomerase activity. The percentage of positive cells of hTERT protein declined with time after hTERT AS PS-ODN treatment, which appeared after the decline of hTERT mRNA. There was no difference in telomerase activity, hTERT mRNA and protein levels between hTERT sense phosphorothioate oligodeoxynucleotide (S PS-ODN) and the control group. The cell viability decreased with time after hTERT AS PS-ODN combined with TNF-alphatreatment. The percentage of apoptosis increased with time after hTERT AS PS-ODN combined with TNF-alpha treatment. There was no difference in cell viability and the percentage of apoptosis between hTERT S PS-ODN and the control group.

CONCLUSIONhTERT AS PS-ODN can significantly inhibit telomerase activity by downregulating the hTERT mRNA and protein expression, and inhibition of telomerase with hTERT antisense can enhance TNF-alpha-induced apoptosis of PC3 cells.

Actins ; metabolism ; Apoptosis ; drug effects ; Cell Line, Tumor ; DNA Primers ; Humans ; Male ; Oligodeoxyribonucleotides ; pharmacology ; Prostatic Neoplasms ; RNA, Messenger ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Telomerase ; antagonists & inhibitors ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; pharmacology

The management of asthma focuses on the reduction of airway inflammation accompany with symptomatic care after recognition. Glucocorticosteroid is the most important drug to reduce airway inflammation, and it has been used inhaled, orally and systemically. New knowledge about the pathogenesis of allergy and asthma has made the development and clinical trial of target or immunomodulator therapy. It includes cytokine, cytokine blockers, specific cytokine receptor blocker, and immunostimulatory oligodeoxynucleotides. These agents are thought to hold the promise for more beneficial outcomes in the future, although it showed limited therapeutic benefits only for patients, especially intractable or severe asthma, until now.
Asthma ; Humans ; Hypersensitivity ; Immunomodulation ; Inflammation ; Oligodeoxyribonucleotides ; Receptors, Cytokine

By using decoy-oligodeoxynucleotides (decoy-ODNS) technique, the effects of Stathmin gene on the proliferation and differentiation of in vitro cultured precartilainous stem cells (PSCs) were investigated. The Stathmin decoy-ODNs were transfected into PSCs in rats by using gene transfection technique. Under the induction of cortisol (1 micromol/L), electrophoretic mobility shift assay was used the inhibitory effects of decoy-ODNS on Stathmin gene. MTT and cytometry were used to test the cell proliferation. The expression of collagen II and V and Stathmin protein was detected by using Western blot. The results showed that Stathmin decoy-ODNs inhibited the Stathmin activity in a dose-dependent manner. When the concentration of decoy-ODNs was 10 times of standard concentration, the proliferation of PSCs was obviously suppressed and the differentiation happened. Compared to the control group, the difference was significant (P<0.05). It was concluded that decoy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.
Cartilage/*cytology ; Cell Differentiation/*drug effects ; Cell Proliferation/drug effects ; Cells, Cultured ; Oligodeoxyribonucleotides/genetics ; Oligodeoxyribonucleotides/*pharmacology ; Rats, Sprague-Dawley ; Stathmin/*genetics ; Stathmin/pharmacology ; Stem Cells/*cytology

To search the effective antisense oligonucleotide that inhibit the growth of Herpes simplex virus type 1 (HSV-1), six kinds of phosphorothioate oligodeoxynucleotides (S-ODNs) were synthesized and the antiviral activity was assessed by measuring cytopathic effect in Vero cells infected with HSV-1. Of the three dodecamer S-ODNs, cornplernentary to the translation initiation site of IE2 (AS2) and scrambled S-ODN (AS1) showed more significant antiherpetic activity than AS4 complementary to the IE4. Accordingly, the antiviral effect of dodecamer S-ODN was not specific. In contrast to the no inhibitory effect of sense strand S-ODN of ICP8 (AS6), two S-ODNs complementary to the translation initiation site of ICP8 (AS3) and that of IE1 (AS5) showed potent antiherpetic activity assessed in vitro HSV-1 virus yield assay. Antiherpetic effect of AS3 was decreased in proportion to the addition of AS6. The synthesis of viral protein ICPS and IE1 were inhibited in AS3 and AS5 treated HSV-1 infected Vero cells, respectively. These findings suggest that antiherpetic effect of AS3 is specifically mediated by targeting ICPS. S-ODNs had no effect on Vero cell viability. The data suggest that the 19-mer S-ODNs may be effective in antiviral chemotherapy.
Drug Therapy ; Herpes Simplex* ; Herpesvirus 1, Human ; Oligodeoxyribonucleotides ; Phosphorothioate Oligonucleotides* ; Simplexvirus* ; Vero Cells

Atopic dermatitis is a chronic, relapsing, inflammatory skin disease, with genetic and environmental backgrounds. Successful management of atopic dermatitis requires a multipronged approach. Management should compromise of a disease-adapted treatment which combines adjuvant basic therapy, symptomatic relief and, if needed, anti-inflammatory treatment and the identification and avoidance of trigger factors. Topical calcineurin inhibitors are also considered to be good alternatives for the long-term control of severe atopic dermatitis. Also, new therapies such as immunomodulatory drugs, biologics, anti-IgE therapy, and CpG oligodeoxynucleotides (ODN) are considered to be effective in treatment of atopic dermatitis.
Antibodies, Anti-Idiotypic ; Biological Agents ; Calcineurin ; Dermatitis, Atopic ; Oligodeoxyribonucleotides ; Skin Diseases

OBJECTIVEThe present study was to evaluate the effects of nuclear factor of kappa B decoy oligodeoxynucleotides on murine multiple myeloma models.

METHODSThe severe combined immunodeficient mice were injected subcutaneously with RPMI-8226 myeloma cells. When tumors became measurable, the mice were divided into 2 treatment groups who respectively received 5 µg/g or 10 µg/g liposome-NF-κB decoy ODN compounds, and one control group was selected; the control group received 10 µg/g liposome-NF-κB mutant decoy ODN compounds, twice per week for 4 weeks. The mice were killed when they died or the tumor diameter became >2 cm.

RESULTSThe liposome-NF-κB decoy ODN could efficiently suppress NF-κB DNA binding activity and inhibited the expression of IL-6. As compared with the control group, the two liposome-NF-κB decoy ODN-treated groups showed more remarkably survival time and smaller tumor volume.

CONCLUSIONIn vivo transfection of NF-κB decoy ODN may provide a new therapeutic strategy for multiple myeloma.

Animals ; DNA ; Disease Models, Animal ; Genetic Therapy ; Interleukin-6 ; Liposomes ; Mice ; Multiple Myeloma ; Oligodeoxyribonucleotides ; Transfection

OBJECTIVETo investigate the efficacy of cryoablation combined with CpG ODN in the treatment of murine transplanted colon carcinoma.

METHODSColon carcinoma model was established by subcutaneously inoculating CT26 cells into the right flank in BALB/c mice. The tumor-bearing mice were randomly divided into 4 groups:the group of PBS injected in peritumoral area, the group of cryoablation, the group of cryoablation combined with CpG ODN, the group of CpG ODN injected in peritumoral area. The tumor size changes were measured. Serum levels of interleukin (IL)-12 and interferon-gamma(IFN-gamma) were assayed by ELISA. The rates of CD3(+)CD4(+)T, CD3(+)CD8(+)T lymphocytes in serum were counted with flow cytometry. Mice in the cryoablation group and the combined group with tumor regression were re-challenged with CT26 cells.

RESULTSThe survival time of cryoablation group and combined therapy group were (80.3 + or - 5.4) days and (83.8 + or - 5.5) days, respectively, longer than (53.7 + or - 3.7) days in PBS group and (51.5 + or - 6.8) days in CpG ODN group(all P<0.05). The suppress rates of tumor cells in cryoablation group and combined therapy group were 83.8% and 86.2% respectively. After 20 days following treatment, CD3(+)CD4(+)T/CD3(+)CD8(+)T ratio and the concentrations of IL-12 and IFN-gamma in mice serum of cryoablation group and combined therapy group were higher than those in PBS group and CpG ODN group(all P<0.05). No significant difference was found in CD3(+)CD4(+)T/CD3(+)CD8(+)T ratio between cryoablation group and combined therapy group(P>0.05). However, the concentrations of IL-12 and IFN-gamma in combined therapy group were higher than those of cryoablation group(all P<0.05). After re-challenging, tumor formation rate in the cryoablation combined with CpG ODN group was 16.7%, significantly lower than that in the cryoablation group(83.8%)(P<0.05).

CONCLUSIONCryoablation combined with CpG ODN can increase antitumor immune response in mice, and therefore can decrease the tumor formation when re-challenged with CT26 cells.

Animals ; Colonic Neoplasms ; therapy ; Cryosurgery ; Female ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Oligodeoxyribonucleotides ; therapeutic use

To optimize the operating variables that affect the transfection of antisense oligodeoxyribonucleotide (AS-ODNs) by insonated gas-filled lipid microbubbles, SF6-filled microbubbles were prepared by sonication-lyophilization method. An AS-ODNs sequence and a breast cancer cell line SK-BR-3 were used to define the various operating variables determining the transfection efficiency of SF6-filled microbubbles. Three levels of mixing speed, different durations of mixing and various delay time before ultrasound were examined, separately. Transfection efficiency was detected by fluorescence microscopy. Transfection results with and without incubation of AS-ODNs and microbubbles before mixing cells were compared. From the results, there is no significant difference between the transinfection efficiency with or without incubation of AS-ODNs and microbubbles before mixing cells. AS-ODNs transfection efficiency showed an increasing trend with mixing speed and mixing duration, but there is a negative relationship with delay time before ultrasound. The optimum parameters for AS-ODNs transfection by SF6-filled microbubbles were found at a mixing speed of 40-50 r x min(-1) for 30-60 s with less than 60 s delay before ultrasound. For a successful transfection, long time of incubation with gene is essential for normal nonviral vectors such as liposomes or cationic lipid-polymer hybrids, because these vectors depend on endocytosis and membrane fusion to realize transfection. Unlike liposomes and cationic lipid-polymer hybrids, gas-filled lipid microbubbles depend on sonorporation effect to realize transfection. Therefore, the incubation of gene and microbubbles before mixing cells may not be necessary. Ultrasound-mediated AS-ODNs transfection enhanced by gas-filled lipid microbubbles represents an effective avenue for gene transfer.
Cell Line, Tumor ; Green Fluorescent Proteins ; Humans ; Microbubbles ; Oligodeoxyribonucleotides, Antisense ; genetics ; Sulfur Hexafluoride ; Transfection ; methods ; Ultrasonics

Desensitization, Immunologic ; methods ; Oligodeoxyribonucleotides ; therapeutic use ; Toll-Like Receptor 9 ; therapeutic use