In vitro lipofectamine mediated NF-κB decoy oligodeoxynucleotides transfection of Kupffer cells / 中华普通外科杂志

Objective To study the transfection effects of nuclear factor-KappaB(NF-κB)decoy oligodeoxynucleotides(ODN) to Kupffer cells (KCs) mediated by lipofectamine,and investigate it's suppression effects on KCs activation. Methods Twenty-four Wistar rats were divided into three groups (n=8).(1)Control group,in which the normal KCs were isolated.(2)LPS group,in which 1 ms/L LPs was added to the culture system.(3)NF-κB decoy ODN group,in which KCs were transduced with NF-κB decoy ODN (4μg×105KCs)prior to LPS stimulation.The transfection efficiency Was assayed,and the phagocytosis function,NF-κB(P65) translocation,CD40 mRNA expression of KCs were also detected respectively. Results Kupffer cells were obviously activated after LPS stimulation.the phagocytosis function was reinforced.the activity of NF-κB transloeated from cytoplasm into nucleus was obviosly increaced.The co-stimulatory molecules expression(CD40 mRNA)significantly increased compared with control group(t=4.01,P<0.01).NF-κB decoy oligodeoxynucleotides can efficiently transfected into KCs mediated by lipofectamine,which can obviously suppress KCs activation,and downregulate the expression of downstream gene(compared with LPS group,t=4.89,P<0.01). Condusion NF-κB decoy ODN can efficiently transfect into KCs and inhibit it's activation.

Effect of nuclear factor-kappa B decoy oligodeoxynncleotides on respiratory function and cytokine expression after severe lung contusion in rabbits / 中华创伤杂志

Objective To explore the effect of nuclear factor-kappa B (NF-κB) decoy oligode-oxynucleotides (ODN) on respiratory function and expressions of IL-1β and IL-13 in serum following se-vere lung contusion in rabbits. Methods A total of 40 New-Zealand rabbits were randomly divided into four groups, ie, severe lung contusion group (Group A, n=12), severe lung contusion with NF-κB scrambled decoy ODN intervention group (Group B, n=12), severe lung contusion with sense NF- B de-coy ODN intervention group (Group C, n=12) and normal control group (Group D, n =4). After the contusion model was set up, the sense and scrambled NF-κB decoy ODN were infused into the rabbits via the jugular veins in different groups, with 20 g per experimental rabbit. After contusion, respiratory fre-quency, tidal volume, airway pressure, respiration flow rate curve and end expiration nitric oxide concen-tration were detected at 1, 2, 3 and 4 hours. The expressions of IL-1β and IL-13 in serum were observed by means of ELISA. Results After sense NF-κB decoy ODN intervention, alveolar ventilation, arteri-al PO_2 and pulmonary compliance were improved, compared with Group A and Group B, with statistical difference (P<0.01). The expression of IL-1β was decreased and that of IL-13 increased after sense NF-κB decoy ODN intervention to the severe lung contusion, compared with Groups A and B, with statis-tical difference (P <0.01). The expression of IL-1β was increased to peak level at 1 hour after contu-sion, which continued to the end of the experiment. While expression of IL-13 was decreased at 1 hour af-ter contusion and reached the minimum level at 4 hours. With intervention with sense decoy ODN, the in-creased expression of IL-1β was down-regulated, but expression of IL-13 remained at high level, with sta-tistical difference compared with Group A and Group B (P < 0.01). Conclusions Intervention with sense NF-κB decoy ODN can significantly protect the respiratory function, reduce the expression of IL-1β and increase expression of IL-13 after severe lung contusion.

Effect of Stathmin Decoy-oligodeoxynucleotides on the Proliferation and Differentiation of Precartilainous Stem Cells / 华中科技大学学报（医学）（英德文版）

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 trans- fection technique. Under the induction of cortisol (1 μmol/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 con- centration, 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 de-coy-ODNs could inhibit the proliferation and promote the differentiation of PSCs by antagonizing Stathmin activity.

Role of Activator Protein-1 in the Transcription of Interleukin-5 Gene Regulated by Protein Kinase C Signal in Asthmatic Human T Lymphocytes / 华中科技大学学报（医学）（英德文版）

In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided int9 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL5 mRNA (0. 8300±0. 0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0. 3050±0. 0208, respectively, P＜ 0.01), while the indexes (23 219.83±1 024.86 and 0. 3425±0. 0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P＜0.01). The indexes (87 107. 41±1 342.92 and 0. 8225±0. 0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P＞0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P＜ 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.

The effect of transcription factor SP1 decoy oligodeoxynucleotides on expression of ? Gal in SV-40-PED cells / 医学研究生学报

Objective:To investigate the role of transcription factor SP1 decoy oligodeoxynucleotides(ODN) on expression of ? Gal in SV-40-PED cells.Methods:Immortalized porcine aortic endothelial cells of the PED line were cultured and transfected with ?1,3galactosyltransferase(?1,3GT) specific decoy ODN.Cells transfected with mismatch ODN was used as negative controls.Twenty-six hours later the cells were collected.The expression of ? Gal was determined with fluorescence microscope and Western blot.The expression of ?1,3GT mRNA was examined by RT-PCR.Results:Fluorescence microscopy observed the decreased fluorescence of ? Gal after decoy ODN transfection.Western blot showed that the average absorbance of the PED cells transfected with decoy ODNs was(48.2?0.9).It is 52.6% of the mock group(P0.05).Conclusion:?1,3GT gene reduce actually occurs following transfection of decoy ODN.Porcine endothelial cells can be the targets of decoy ODN.

The effect of STAT3 decoy oligodeoxynucleotides on the expression of IFI16 in peripheral blood mononuclear cell of patients with systemic lupus erythematosus / 中华风湿病学杂志

Objective To investigate the effect of signal transducer and activator of transcription 3 (STAT3) decoy oligodeoxynucleotides (ODNs) on the expression of IFI16 in the peripheral blood mononuclear cell of patients with systemic lupus erythematosus (SLE).Methods STAT3 decoy ODNs was transfected to PSMC in vitro,and RT-PCR was used to semiquantitatively analyze the IFI16 mRNA in the peripheral blood mononuclear cell (PBMC) before and after the transfection.Results The mean level of IFI16 mRNA in ac- tive SLE group and inactive SLE group were much higher than that of normal control.The difference was sig- nificant (P＜0.05).However,there was no statistical difference between active and inactive SLE group (P＞0.05).After being transfected by STAT3 decoy ODNs,the level of IFI16 mRNA in active and inactive SLE group were markedly decreased comparing with negative controls.The difference was significant (P＜0.05). However,the difference was not significant in normal control (P＞0.05).Conclusions The abnormal overex- pression of IFI16 mRNA in PBMC of patients with SLE suggests that IFI16 may contribute to the pathogenesis of SLE and the overexpression of IFI16 may be mediated through JAK-STAT3 pathway.

Optimizing condition for lipofectin-mediated NF-?B decoy oligodeoxynucleotides transfection into J774.1 cells / 第二军医大学学报

Objective: To search for the optimizing parameters and distribution pattern of NF ?B decoy oligodeoxynucleotides in transfecting J774.1 cells mediated by lipofectin. Methods: With the change of ODNs/lipofectin ratio and transfection time, the uptake rate and mean fluorescence intensity of NF ?B decoy oligodeoxynucleotides in J774.1 cells were measured by flow cytometry to evaluate transfection efficiencies. Intracellular distribution of NF ?B decoy oligodeoxynucleotides was determined with fluorescence microscopy. The lactate dehydrogenase (LDH) activity in the supernatant was assayed to assess the cytotoxicity. Results: The uptake of NF ?B decoy oligodeoxynucleotides into J774.1 cells was significantly improved by lipofectin. In 24 well culture plate, 1 ?g ODNs with 5?g lipofectin ( W/W =1∶5) resulted in the highest transfection efficiency and lower cytotoxicity. The NF ?B decoy oligodeoxynucleotides localized in the nucleus as well as in the cytoplasm following an incubation of 6 h with lipofectin. While NF ?B decoy oligodeoxynucleotides had faint fluorescences in cytoplasm in the absence of lipofectin. Conclusion: lipofectin can enhance the cellular uptake of NF ?B decoy oligodeoxynucleotides in J774.1 cells and alter intracellular distribution of NF ?B decoy oligodeoxynucleotides. Efficiency of transfection is the highest when the ratio of ODNs/lipofectin is 1∶5 for incubation of 6 h.

Inhibitive effect of peritoneal decoy-oligoligomucleotidesnucleotides targeting nuclear factor-?B on expression of inflammatory mediators in macrophages ? cells / 中华创伤杂志

Objective To design and synthesize targeting nuclear factor-?B (NF-?B) decoy-oligodeoxynucleotides (ODNs) and measure their anti-inflammatory actions. Methods Peritoneal macrophages ? (pM?) cells extracted from rats were randomly divided into normal control group, lipopolysaccharides (LPS)-stimulated group (Group A), decoy-ODNs treated group (Group B), non-decoy-ODNstreated group (Group C) and cationic liposome treated group (Group D). Supernatants and cells in the control group and the Group A were collected 1,2,6,12,18 and 24 hours after LPS stimulation. By using cationic liposomes in a ratio of 4:1 in quantity, pM? cells were transfected by decoy-ODNs at concentrations of 2 mg/L,4 mg/L and 8 mg/L respectively and stimulated with LPS 6 hours later. Then, supernatants and cells were collected 8, 12 and 18 hours after transfection. The expression changes and the distribution of p65 were analyzed by immunocytohistochemical method, the protein expressions of tumor necrosis factor ? (TNF?), interleukin-6 (IL-6) and interleukin-10 (IL-10) in the supernatants by enzyme-linked immunosorbent assay (ELISA) and mRNA transcriptions of TNF?,IL-6 and IL-10 by reverse transcriptase polymerase chain reaction (RT-PCR). Results Decoy-ODNs at concentration of 4 mg/L and 8 mg/L could markedly inhibit the expression and transcription of TNF? and IL-6 of pM? cells in a dose-dependent fashion but had a weak inhibitive effect on the expression and transcription of IL-10. Conclusions The targeting NF-?B Decoy-ODNs can suppress the expressions of inflammatory mediators in pM? cells.

Effects of NF-?B “decoy” oligodeoxynucleotides on IL-10 expression in LPS-induced mouse macrophages / 中国病理生理杂志

AIM: To study the effect of NF-?B “decoy”oligodeoxynucleotides on IL-10 expression in LPS-induced mouse macrophages. METHODS: Mouse macrophage cell line J774 1 cells were cultured with LPS and liposome-mediated oligodeoxynucleotides, and the level of IL-10 were measured in culture medium by enzyme linked immunosorbent assay. RNA was extracted from macrophages, and the mRNA expression of IL-10 were observed by RT-PCR. RESULTS: NF-?B “decoy”oligodeoxynucleotides did not inhibit the expression of IL-10 in LPS-induced macrophage, and did not reduce generation of IL-10. CONCLUSION: NF-?B decoy ODNs did not depress the mRNA expression of IL-10. NF-?B may not be the critical transcription factor in transduction mechanism of IL-10.