OBJECTIVEObserving the time course and establishing the model of kappaB-decoy oligodeoxynucleotides (rcB-decoy) inhibiting the activity of NF-kappaB in the PC12 cells.

METHODSPC12 cells cultivating in the 6 wells plate were divided into 3 groups, experimental group: adding kappaB-decoy complex (6 microg DNA/well), the control group: adding scrambled-decoy complex, the normal group: adding lipid-Lipofectamine 2000, transfer and cultivate 48 h, then lipopolysaccharide (LPS, 200 ng/ml) was added in the cells for 0.5-4 h. The immunocytochemistry and Western blot were used to measure the expression or the activity of NF-kappaB in PC12 cells.

RESULTSIn PC12 cells, compared with normal group, the expression of NF-kappaB enhanced obviously with the time of the stimulation of LPS in scrambled-decoy treated control group (P < 0.01), in 2-4 h the level reached the peak; the expression of NF-kappaB showed the stable level with the time of the stimulation of LPS in kappaB-decoy treated experimental group, compared with the control group, the expression levels were obviously lower than the respective time point of control groups (P < 0.01).

CONCLUSIONkappaB-decoy could reduce the expression of NF-kappaB in the normal PC12 cells and inhibit the activity of NF-cB in the pathologic PC12 cells.

Animals ; Cells, Cultured ; Lipopolysaccharides ; pharmacology ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Oligodeoxyribonucleotides ; pharmacology ; PC12 Cells ; Rats

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

OBJECTIVETo study the maturation effect of CpG2006 and phosphodiester oligonucleotides on leukemia-derived dendritic cells.

METHODSLeukemia cells K562/A02 were induced into dendritic cells by rhGM-CSF and rhIL-4. After 7 days induction, the cell-morphology was observed, the immunophenotype of cells was detected by flow cytometry and the cell function was evaluated by allogeneic mixed lymphocyte reactions, CTL responses and secretion of IL-12 and IL-6. Then a CpG oligonucleotide CpG2006, two synthetic bacterial phosphodiester oligonucleotides A-ODN and T-ODN were added to these leukemia-derived DCs. Three days later, the DCs were re-detected by the above-mentioned methods.

RESULTSAfter induced by CpG2006, A-ODN or T-ODN, the leukemia-derived DCs with typical dendritic morphology were increased. The expressions of CD83, HLA-DR and CD86 were (65.5 +/- 8.4)%, (32.0 +/- 4.3)% and (18.6 +/- 3.2)% respectively in day 7 leukemia-derived DCs, raised to (88.9 +/- 3.6)%, (53.9 +/- 3.2)% and (39.9 +/- 7.3)% respectively after exposing CpG2006 for 3 days; increased to (97.0 +/- 5.3)%, (63.9 +/- 7.3)% and (40.2 +/- 7.4)% respectively after treated by A-ODN; and further increased to (93.26 +/- 4.65)%, (58.3 +/- 5.6)% and (36.2 +/- 6.8)% respectively after treated by T-ODN. These results was markedly different than unaffected cells did. These DCs induced by the above-mentioned three oligonucleotides could upregulate significantly the capacity for stimulating allogeneic T cells. They could also induce CTL to generate specific cytotoxic activity against K562/A02 cells. The secretion of IL-6 and IL-12 was increased remarkably.

CONCLUSIONCpG2006, as well as two phosphodiester oligonucleotides can induce leukemia-derived DCs maturation.

Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Dendritic Cells ; cytology ; drug effects ; Humans ; K562 Cells ; Oligodeoxyribonucleotides ; pharmacology ; Oligonucleotides ; pharmacology

Animals ; Inflammation ; chemically induced ; Lung ; drug effects ; Oligodeoxyribonucleotides ; pharmacology ; Pulmonary Alveoli ; drug effects ; pathology ; Silicon Dioxide ; adverse effects

To investigate the specific anti-leukemia effect of cytotoxic T lymphocytes (CTLs) induced by dendritic cells (DCs) activated by exosomes alone or in combination with CpG ODN in vitro and the feasibility of exosomes as remedial vaccine, the DCs induced from normal volunteer PBMNCs were divided into 7 groups. Three groups of them were added with the exosomes: Kexo (exosomes derived from K562 cells) or DCexo (exosomes derived from DCs induced from K562 cells) or FTexo (exosomes derived from DCs induced from K562 cells and pulsed by freeze-thawing antigen of K562 cells) as experimental groups (Kexo, DCexo and FTexo). The other three groups were added with CPG ODN while added the exosomes (Kexo, DCexo and FTexo), and were used as experimental groups also (Kexo+CpG, DCexo+CpG and FTexo+CpG). The seventh group DCs was added with nothing as blank control. These DCs above mentioned were cultured continuously for 72 hours. The T lymphocytes were co-cultured with DCs for another 72 hours to generate CTL. Then, the killing effects of them on K562 cells were determined by MTT assay. The results showed that all experimental groups pulsed by exosomes displayed stronger killing effect, compared with control group (p<0.05). DCexo and FTexo displayed stronger killing effect too, compared with Kexo (p<0.05). CPG ODN as an adjuvant could enhance the killing effect (p<0.05). It is concluded that the special killing effect on K562 cells can be induced by exosomes, CPG ODN as an adjuvant can enhance the killing effect. Exosome is hopeful as a remedial vaccine to be used for the leukemia therapy.
Adjuvants, Immunologic ; pharmacology ; Cancer Vaccines ; immunology ; Dendritic Cells ; immunology ; Exosomes ; immunology ; Humans ; K562 Cells ; Oligodeoxyribonucleotides ; immunology ; T-Lymphocytes, Cytotoxic ; immunology

To study the effect of VEGF fully phosporothioated antisense oligodeoxynucleotide (VEGF-ASODN) on VEGF expression in acute monocyte leukemic cell line U937 in vitro, U937 cells were incubated with VEGF-ASODN at concentrations of 10, 20 and 30 micro mol/L or scrambled sequence as compared with negative control. The expression of VEGF mRNA was measured by semi-quantitative RT-PCR. The expression of VEGF protein was measured by Western blot. The result showed that VEGF-ASODN had obviously inhibitive effect on expression of VEGF in U937 cell, as compared with scrambled sequence and negative control (P < 0.05). Scrambled sequence group had no significant difference compared with negative control group (P > 0.05). It is concluded that the expressions of VEGF mRNA and protein in leukemic cell line U937 are down-regulated by VEGF-ASODN.
Humans ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; RNA, Messenger ; analysis ; U937 Cells ; Vascular Endothelial Growth Factor A ; analysis ; antagonists & inhibitors ; genetics

OBJECTIVETo observe the inhibitory effect of oligodeoxynucleotides (ODN) on nuclear translocation and nuclear binding activity of nuclear factor (NF)-kappaB in THP-1 cells.

METHODSOligodeoxynucleotides were transfected via liposome into THP-1 cells followed by stimulation of the cells with lipopolysaccharide (LPS). Immunocytochemistry, electrophoretic mobility shift assay (EMSA) and reverse transcription (RT)-PCR were performed to detect the nuclear translocation and nuclear binding activity of NF-kappaB.

RESULTSImmunocytochemical results showed that after LPS stimulation of the ODN-transfected cells, NF-kappaB expression was still localized in cytoplasma. EMSA demonstrated inhibited nuclear binding activity of NF-kappaB in the ODN-transfected cells, and ODN inhibited the mRNA expression of tumor necrosis factor (TNF)-alpha, a NF-kappaB-associated inflammatory factor, as shown by RT-PCR.

CONCLUSIONODN can inhibit the nuclear translocation and binding activity of NF-kappaB in THP-1 cells, whereby the transcription and expression of the related inflammation factor genes is suppressed, which shed light on a new solution for clinical treatment of acute pancreatitis.

Cell Line ; Humans ; Lipopolysaccharides ; Macrophages ; cytology ; drug effects ; metabolism ; NF-kappa B ; metabolism ; Oligodeoxyribonucleotides ; pharmacology ; Transcription Factors ; metabolism ; Transfection

OBJECTIVE: To study the effects of oligodeoxynucleotides complementary Stat3 on apoptosis in laryngeal carcinoma Hep-2 cell. METHOD: Oligodeoxynucleotides complementary Stat3 was designed, which was transferred into laryngeal carcinoma Hep-2 cell by lipofection. Expression of Stat3 and p-Stat3 were detected by Western blot and PCR. MTT was used to observe the growth-inhibiting ratio. DNA ladder, AO/EB and FCM were used to observe the apoptosis of laryngeal carcinoma cell Hep-2 in vitro. RESULT: Western blot and PCR results demonstrated that oligodeoxynucleotides complementary Stat3 could significantly inhibit the expression of Stat3 and p-Stat3 in Hep-2 cell. MTT results showed that it could significantly suppress the growth of Hep-2 cell. The DNA ladder, AO/EB and FCM results showed it could inhibit the expression of Stat3 and induce the apoptosis of Hep-2 cell in a concentration-dependent manner. CONCLUSION Oligodeoxynucleotides complementary Stat3 could induce the apoptosis and suppress cell proliferation in laryngeal carcinoma Hep-2 cell.
Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Oligodeoxyribonucleotides ; genetics ; pharmacology ; STAT3 Transcription Factor ; genetics ; Transfection

OBJECTIVETo investigate the effect of S-phase kinase-associated protein 2 antisense oligodeoxynucleotide (Skp2 ASODN) on the growth and proliferation of gastric carcinoma SGC-7901 cells and its mechanism.

METHODSThe Skp2 oligodeoxynucleotides (ODNs) were embedded in cationic liposome Lipofectamine 2000 reagent and transfected into SGC-7901 cells. The cell growth and proliferation were observed with light microscopy and MTT assay. Cell cycle was measured by flow cytometry. The expression levels of Skp2 and p27 mRNA were detected by reverse transcription-polymerase chain reaction. The expression levels of Skp2 protein and its substrate p27 protein were detected by Western blot.

RESULTAfter treatment with Skp2 ASODN, the growth and proliferation of SGC-7901 cells were inhibited in a dose-dependent manner with a peak value at 48 h. The inhibition rate of 200 nmol/L group at 48 h was 42.4 % (P<0.01). In cell cycle study the percentage of S phase cells in 200 nmol/L group was significantly higher than that in normal control group (P<0.05). Both Skp2 mRNA and its protein levels in 200 nmol/L group were significantly lower than those in control group and in Skp2 nonsense oligodeoxynucleotide (Skp2 NSODN) group (P<0.05). However, p27 mRNA level remained unchanged although its protein level was significantly higher than that in control group and NSODN group (P<0.05).

CONCLUSIONSkp2 ASODN can inhibit the growth and proliferation of SGC-7901 cells, which may be mediated by interfering with ubiquitin-proteosome pathway and cell cycle regulation.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Humans ; Oligodeoxyribonucleotides, Antisense ; genetics ; pharmacology ; S-Phase Kinase-Associated Proteins ; genetics ; pharmacology ; Stomach Neoplasms ; pathology ; Transfection

Cationic PLG nanoparticles and liposome were prepared and used as package molecules to pack up pUC18-CpG. The effects of the packed pUC18-CpG on the cellular and humoral immune responses were detected in the mice that were inoculated with pig paratyphoid vaccine. The results showed that compared with the control, the amount of IgG and the titre of specific antibody were significantly increased in the sera of mice immunized with the CpG plasmid entrapped by cationic PLG nanoparticles; the proliferation and induced IL-2 bioactivity of lymphocytes were significantly enhanced in the spleen of the immunized mice; the stimulatory effect of cationic PLG nanoparticles was similar to or stronger than that of cationic liposome. These indicated that cationic PLG nanoparticle could be employed as an effective package molecule to promote the immunostimulatory effect of pUC18-CpG.
Adjuvants, Immunologic ; pharmacology ; Animals ; Immunoglobulin G ; blood ; Interleukin-2 ; blood ; Liposomes ; Male ; Mice ; Mice, Inbred BALB C ; Nanostructures ; Oligodeoxyribonucleotides ; pharmacology ; Swine ; Typhoid-Paratyphoid Vaccines ; immunology