Effect of intranasal treatment with Toll-like receptor 9 ligand CpG oli-godeoxynucleotides on airway inflammation in mice with allergic com-bined airway disease / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the therapeutic effect of intranasal administration of CpG oligodeoxynucleotides (CpG-ODN), compared with intradermal administration, on lower airway inflammation in ovalbumin (OVA)-induced al-lergic combined airway disease (ACAD) mouse model.METHODS: Totally 30 female BALB/c mice aged from 6 to 8 weeks were randomly divided into control group , allergic rhinitis model group (AR group), ACAD group, ACAD intrana-sally treated with CpG-ODN group (CpG i.n.group) and ACAD intradermally treated with CpG-ODN group (CpG i.d. group).The mice were sensitized and challenged with OVA .Treatment with CpG-ODN was also performed during chal-lenge, either intranasally or intradermally .Immunologic variables and nasal symptom were studied .RESULTS:Compared with CpG i.d.group and ACAD group, the percentage of eosinophils from bronchoalveolar lavage fluid (BALF), the levels of Th2 cytokine production in BALF and supernatants of cultured splenic lymphocytes , OVA-specific IgE from blood , peri-bronchial inflammation score in the lung , and nasal symptoms were significantly reduced in CpG i .n.group.CONCLU-SION:Allergic rhinitis treated by CpG-ODN has a significant improvement on lower airway inflammation in ACAD mouse model;and it may be more effective when administrated intranasally than intradermally .

Effect of Total Glucosides of Paeony on the Expression of Toll-like Receptor 9 in Peripheral Blood B Cells in the Patients with Systemic Lupus Erythematosus / 医药导报

Objective To investigate the effect of total glucosides of paeony ( TGP ) on the expression of Toll-like receptor 9 (TLR9) in peripheral blood B lymphocytes of the patients with systemic lupus erythematosus (SLE). Methods Sixty SLE patients and thirty healthy volunteers were enrolled, peripheral blood mononuclear cells ( PBMC) were isolated from blood samples and divided into 4 groups, which were incubated with CpG-ODN(final concentration was 1 μmol·L-1), CpG-ODN+TGP ( TPG final concentration was 1í10-4 mol·L-1 ) ,TGP and RPMI medium ( as the blank control group) for 48 hours, respectively. FLA ( Flow cytometry analysis) was used to detect the expression of TLR9 on peripheral blood B lymphocytes after incubated. Results ①In the health people, TLR9 expression in the group of CpG-ODN was(9. 10±2. 12) %, which was higher than the blank control group(4. 96±2. 11) % (P<0. 01).②In the SLE patients, the TLR9 expression in the group of CpG-ODN was(14. 86±3. 42)% , which was significantly higher than the blank control group(9. 20±3. 43) %(P<0. 01). The TLR9 expression in the group of CpG-ODN+TGP was (11. 95±3. 63)%, which was lower than that in the group of CpG-ODN (P<0. 05). The TLR9 expression in the group of CpG-ODN with lightly active degree SLE patients was (10. 74±3. 17)%, which was higher than the blank control group(5. 19±2. 05) % (P<0. 01). For SLE from the moderately to vigorously active degree, the expression of TLR9 in the group of CpG-ODN(16. 51±1. 72) % was higher than the blank control group(10. 80±2. 37) %(P<0. 01), but that in the group of CpG-ODN+TGP (13. 59±2. 58) % was lower than the group of CpG-ODN (P<0. 01). Conclusion Our data indicate that TGP antagonize upregulation effect of CpG-ODN on the expression of TLR9 in peripheral blood B lymphocytes.

Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-gamma-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats / 中国病理生理杂志

[ABSTRACT]AIM:ToevaluatetheeffectsofantisenseTGF-β1oligodeoxynucleotide(ASTGF-β1)ontheex-pression of TGF-β1 , deposition of extracellular matrix ( ECM) and the neointima formation in the arteries after balloon inju-ry.METHODS:The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed.At the same time, sense TGF-β1 oligodeoxynucleotide ( S TGF-β1 ) with the base sequence complement to AS TGF-β1 was synthesized as a control . The oligodeoxynucleotides were introduced into in vivo and in vitro experiments , respectively .RESULTS:The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner , and S TGF-β1 did not have the same effect.Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was ob-served.Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis.Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs . Fibronectin ( FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (0.01~1 μmol/L)-dependent manner .AS TGF-β1 significantly increased the mRNA expression of contractile marker SM 22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla , especially at the concentration of 0.01μmol/L and 0.1 μmol/L.After treatment with AS TGF-β1 (90 μg· kg-1 · d-1 ) for 28 d, the neointima formation was significantly inhibited , and the area ratio of intima/media was markedly decreased by 68% compared with untreated group , but S TGF-β1 had no effect on neointimal formation .CONCLUSION:The AS TGF-β1 specifically inhibits the pro-tein expression of TGF-β1 in the VSMCs derived from injured arteries .Moreover , it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN .Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury .The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the altera-tion of VSMC phenotype after balloon injury .

Reversal of multiple drug resistance in Escherichia coli by blocking CTX-M through antisense phosphothioate oligodeoxynucleotides / 中华微生物学和免疫学杂志

Objective To investigate the effects of antisense phosphothioate oligodeoxynucleotides (AS-ODNs) W086 on drug-resistant gene CTX-M expression in Escherichia coli producing extended-spectrum β-lactamases(ESBLs).Methods AS-ODNs liposome was introduced into the purpose bacteria B052.The total colony forming unit(CFU) was counted.The bacteria growth curve was drawn by microplate reader.The inhibition effects of AS-ODNs on the expressions of drug-resistant gene CTX-M were observed by RT-PCR in B052.The minimal inhibitory concentration(MIC)was determined by fluid dilution method.Results significant growth inhibition of cells treated with W086 was observed as compared with those in cells in control treated bacteria.The number of B052 colonies significantly decreased in all W086 treated groups in a concentration dependent manner (P ＜ 0.05),while CFU of B052 was not influenced in simple liposome group,simple W086 group and controlled chain group.The expression of CTX-M was selectively inhibited.Conclusion Efficiently and specificly blocking expression of CTX-M mRNA,AS-ODNs reverses the multiple drug resistance of B052.It indicates that AS-ODNs provides a new viable strategy to reverse antibiotic resistance problem.

Role of ATM kinase in the effect of CpG ODN7909 on X-ray-induced G2/M phase arrest and apoptosis in A549 cells / 中华放射医学与防护杂志

Objective To investigate the relationship between the effect of unmethylated cytosine phosphate-guanine oligodeoxynucleotide ( CpG ODN ) 7909 on X-ray-induced G2/M phase arrest and apoptosis and the phosphorylation of ATM kinase.Methods Human lung adenocarcinoma A549 cells were randomly classified into five groups,control,CpG group,irradiation group,CpG + irradiation group and ATM siRNA + CpG + irradiation group.Cell survival fraction was evaluated by clonogenic assay.Cell cycle and apoptosis were analyzed by flow cytometry.The expressions of ATM,checkpoint kinase-2(Chk2) and p53 were detected with Western blot.Results Compared to the situation under X-ray irradiation alone,decreased cell clonogenic survival,prolonged G2/M arrest,and increased cell apoptosis were observed after the combination treatment with CpG ODN7909 and X-rays ( t =13.41,17.32 and 7.71,P ＜ 0.05).Moreover,the phosphorylations on ATM,Chk2,and p53 were increased in the irradiated A549 cells that had been pre-treated with CpG ODN7909.After ATM siRNA interfering,abrogation of G2/M arrest,reduction of apoptosis,and decrease of Chk2 and p53 phosphorylation were found in A549 cells treated with CpG ODN7909 and X-rays ( t =26.84,2.98,47.24 and 67.47,P ＜0.05).Conclusions CpG ODN7909 can enhance the X-ray-induced phosphorylation of ATM kinase in human lung adenocarcinoma cells in vitro,which might be involved in regulating G2/M phase arrest and apoptosis.

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.

The change of regulatory T cells of PBMC and the effects of CpG ODN in patients with lung cancer / 中国免疫学杂志

Objective:To study the change of regulatory T cells in PBMC and the effects of CpG ODN in patients with lung cancer.Methods: Lymphocytes isolated from blood of 30 patients with lung canaer and 30 healthy volunteers were analyzed for the proportion of CD4 ~+CD25~+ T cells by flow cytometry.The mRNA expression of Foxp3 was detected by real-time PCR and the levels of TGF-β and IFN-γ were tested by ELLSA.The PBMC of 30 patients with lung cancer were randomly divided into treated group(CpG ODN 2006)and placebo group(CpG ODN 1612).The proportion of CD4 ~+ CD25~+ T cells,the mRNA expression of Foxp3 and the levels of TGF-β and IFN-γ were compared before and after treatment.Results:Tbe proportion of CD4~+ CD25~+ T cells,the mRNA expression of Foxp3 and the levels of TGF-β in patients with lung cancer were higher than those in healthy volunteers,but there was no significant difference in such parameters among subgroups of pathologic classification and clinical stage patients.The proportion of CD4 ~+ CD25~+ T cells, the mRNA expression of Foxp3 and the levels of TGF-β were dropped in CpG ODN 2006 group after treatment.There was no significant difference in these parameters before and after treatment in CpG ODN 1612 group.Conclusion: The proportion of CD4~+ CD25 ~+ T cells, the mRNA expression of Foxp3 and the levels of TGF-β of PBMC in lung cancer patients are higher than those in healthy volunteers.Treating by CpG ODN 2006 could down-regulate the proportion of CD4~+ CD25~+ Foxp3~+ regulatory T cells and the levels of TGF-β of PBMC from the patients with lung cancer.

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.

The Effects of Nuclear Factor-kappa B Decoy Oligodeoxynucleotide on Lipopolysaccharide-Induced Direct Acute Lung Injury / 결핵및호흡기질환

BACKGROUND: The pathophysiologic mechanisms of early acute lung injury (ALI) differ according to the type of primary insult. It is important to differentiate between direct and indirect pathophysiologic pathways, and this may influence the approach to treatment strategies. NF-kappa B decoy oligodeoxynucleotide (ODN) is a useful tool for the blockade of the expression of NF-kappa B-dependent proinflammatory mediators and has been reported to be effective in indirect ALI. The purpose of this study was to investigate the effect of NF-kappa B decoy ODN in the lipopolysaccharide (LPS)-induced direct ALI model. METHODS: Five-week-old specific pathogen-free male BALB/c mice were used for the experiment. In the preliminary studies, tumor necrosis factor (TNF)-alpha, interleukine (IL)-6 and NF-kappa B activity peaked at 6 hours after LPS administration. Myeloperoxidase (MPO) activity and ALI score were highest at 36 and 48 hours, respectively. Therefore, it was decided to measure each parameter at the time of its highest level. The study mice were randomly divided into three experimental groups: (1) control group which was administered 50 microliter of saline and treated with intratracheal administration of 200 microliter DW containing only hemagglutinating virus of Japan (HVJ) vector (n=24); (2) LPS group in which LPS-induced ALI mice were treated with intratracheal administration of 200 microliter DW containing only HVJ vector (n=24); (3) LPS+ODN group in which LPS-induced ALI mice were treated with intratracheal administration of 200 microliter DW containing 160 microgram of NF-kappa B decoy ODN and HVJ vector (n=24). Each group was subdivided into four experimental subgroups: (1) tissue subgroup for histopathological examination for ALI at 48 hours (n=6); (2) 6-hour bronchoalveolar lavage (BAL) subgroup for measurement of TNF-alpha and IL-6 in BAL fluid (BALF) (n=6); (3) 36-hour BAL subgroup for MPO activity assays in BALF (n=6); and (4) tissue homogenate subgroup for measurement of NF-kappa B activity in lung tissue homogenates at 6 hours (n=6). RESULTS: NF-kappa B decoy ODN treatment significantly decreased NF-kappa B activity in lung tissues. However, it failed to improve the parameters of LPS-induced direct ALI, including the concentrations of tumor necrosis factor-alpha and interleukin-6 in BALF, myeloperoxidase activity in BALF and histopathologic changes measured by the ALI score. CONCLUSION: NF-kappa B decoy ODN, which has been proven to be effective in indirect models, had no effect in the direct ALI model.

The effects of integrin β1 antisense oligodeoxynucleotide on the expression of F-actin in ECV-304 cells exposed to infrasound / 中华物理医学与康复杂志

Objective To study the effect of integrin β1 antisense oligodeoxynucleotide(ASODN)on the expression of F-actin in ECV-304 ceils exposed to infrasound,and to explore the pathway of infrasound signal transfer into the cells.Methods integrin β1 oligodeoxynucleotides were embedded in cationic liposome lipofectamine 2000 reagent and transfected into ECV-304 cells.Laser scanning confoeal microscopv was used to observe F-actin's expression when the cells were exposed to 16 Hz infrasound at 130 dB for 2 h.The cells' average fluorescence was examined using spectrofluorimetric quantification after immunofluorescent staining. Results Among the three false exposure groups,most fluorescein-labelled substances in the cells were in a diffuse state.There were few actin filaments,and they were tenuous and short, without direction.The cells' fluorescence intensities were almost the same.In a group exposed to infrasound,F-actin was thick and long with a longitudinal arrangement.Fluorescence intensities were strong.However,less F-actin expression was observed in the ceils of the ASODN group exposed to infrasound compared with the others similarly exposed.Correspondingly.F-actin expression in the ASODN group exposed to infrasound was almost the same as in the other exposed group.Conclusion F-actin expression can be increased in ECV-304 cells by infrasound exposure,but integrin β1 ASODN may partially inhibit its expression.The extraeellular matrix-integrin-cytoskeleton may be one of the transduetion pathways for infrasound signals into the cells.

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.

Effects of Livin antisense oligodeoxynucleotides on the proliferation and apoptosis of HL60 cells / 基础医学与临床

Objective To investigate effects of Livin antisense oligodeoxynucleotides(ASODN) on the proliferation and apoptosis of human leukemia(HL60) cells.Methods Livin protein on HL60 cells was examined by immunohistochemistry.Specific phosphorothioate antisense oligodeoxynucleotides and missense oligodeoxynucleotides target Livin mRNA were synthesized and transfected into HL60 cells following cationic liposome.The proliferation inhibition of HL60 cells was assessed by MTT.The expression of Livin mRNA was detected by RT-PCR.Transmission electron microscope and TUNEL technology were used to detect the apoptosis and morphologic change.ResultsASODN of 600 nmol/L inhibited the HL60 cell proliferation and the expressions of Livin mRNA.The percentage of apoptosis detected by TUNEL was 38.48%?4.37%.cellar ultrastructure was markedly destroyed by Livin ASODN.A significant difference was found when compared with the control group(P

Bcl-2 Antisense Oligodeoxynucleotides Increase Apoptosis of Lung Cancer Cells Induced by Radiation / 医学研究杂志

Objective To study the effects of Bcl-2 antisense oligodeoxynucleotides(ASODN)on the apoptosis of lung cancer cells induced by radiation in vitro.Methods NCI-H446 lung cancer cell strains were divided into 5 groups:control simple radiation,lipofectin plus radiation,nonsense sqnence radiation and ASODN plus radiation.The cells cultured in five groups were collected at 6h,12h,24h,48h and 72h,with Wright-Giemsa stain,morphology analysis for which was done;the mRNA expression for p53、bcl-2 and PTEN gene was examined by RT-PCR half quantivity and DNA-ploid of the cells in five groups was detected by flow cyfometric method.Results Cell proliferation is obviously restrained and conformation is changed too with the shape crimpled and adherence function decreased obviously after irradiated for 10 Gy dose by the linac;p53 and PTEN expression clearly increased for the combination of Bcl-2 ASODN and bcl-2 mRNA expression clearly decreased.The apoptosis rate after 72 hours among control,pure radiation,lipofectin+radiation,nonsense+radiation and ASODN +radiation grouop is 0.14?0.09,13.17?2.47,11.84?1.76,13.72?1.4,21.26?2.97 respectively,the difference between ASODN combined with radiation grouop and other 4 groups are significant(P

Effects of Tiam 1 antisense oligodeoxynucleotides transfection on the invasive and migratory potentials of gastric cancer cells in vitro and vivo / 中国癌症杂志

Background and purpose:As a regulatory factor of cytoskeletal structure,a close correlation existed between Tiam 1(T lymphoma invasion and metastasis inducing factor 1) and the invasion and metastasis of gastroenteric tumor.We aimed to observe the effects of Tiam 1 antisense oligodeoxynucleotides(Tiam 1 ASODN) transfection on the invasive and migratory potentials of gastric cancer cells in vitro and vivo.Methods:The higher invasive and migratory subgroup(MH) were separated from human gastric cancer cell line MKN-45(M0) by laminin adhesion method in vitro.Tiam 1 ASODN was transfected into MH cells with liposome,and the expression of Tiam 1 mRNA and protein was determined by RT-PCR and ELISA separately.Changes in the invasive and migratory potentials of MH cells in vitro and vivo after transfected with ASODN were observed by Boyden chamber test and inoculation into nude mice respectively.Results:The expression of Tiam 1 mRNA and protein in MH cells after transfected with 0.43 ?mol/L ASODN(0.162?0.018,0.982?0.119) was significantly lower than that of either Liposome-transfected cells(0.789?0.054,1.237?0.108),sense oligodeoxynucleotides(SODN)-Liposome-transfected cells(0.754?0.039,1.234?0.103) or No-transfected cells(0.801?0.065,1.290?0.182)(P

Role of TLR9 in pathogenesis of glomerulonephritis / 吉林大学学报(医学版)

Objective To investigate the role of TLR9 in the glomerulonephritis through observing the changes of Toll-like receptor 9 in the glomerulo nephritis kidney tissue with or without CpG-ODN stimulation .Methods Wistar male rats were randomly divided into normal group(N),nephritis model group(M),CpG-ODN group(CpG) and GpC-ODN group(GpC).The urine samples were collected at 2,4,6 and 8 weeks after treatment,respectively.Blood samples were collected at the end of the last urine sample was collected,and the kidney tissue was collected,then the rats were killed.24 h urine protein was measured by Coomassie light blue technique.Serum album and renal function were determined by serological method.The pathologic changes of kidney were observed by light microscope and NF-?B p65 expression was detected using immunohistochemystry,RT-PCR was performed to detect the expressions of TLR9,INF-? and IL-6 mRNA.Results The expression of TLR9 was lighter in group M,and significantly increased after CpG-ODN stimulation compared with group M.Furthermore,24 h urine protein excretion was markedly increased,serum album was markedly decreased.The histopathologic changes of kidney were more severe.The mesangial cells(MCs) proliferated diffusifully in midrange and wide range,some of the glomeruli formatted cellularity crescent,micrangium loop was limitted,mononuclear macrophile cells were seen in the mesangial region.Conclusion Inflammatory factors mediated by TLR9 can deteriorate the biochemical and histopathologic changes.The immunologic reaction mediated by TLR9 is one of the mechanisms for the glomerulonephtitis' progression.

Association between apoptosis induced in hepatoblastoma HepG2 cells by Cdk7 silencing and decreased pRb and Cdk2 phosphorylation / 中国药理学通报

Aim: To evaluate the effect of Cdk7 silencing on the cell cycle control, the phosphorylation level changes of Cdk2 and pRb in human hepatoblastoma HepG2 cell culture in vitro, and to validate Cdk7 as a novel target for anticancer therapeutics. Method: Levels of Cdk7 and the phosphorylation levels of Cdk2 and pRb were measured by Western-blotting. DNA contents, cell cycle and apoptosis induced by Cdk7 silencing were analyzed by flow cytometry and ultrastructural changes of cells were observed with transmission electron microscopy. Result The phosphorylation levels of pRb and Cdk2 and the levels of Cdk7 decreased in a concentration-dependent manner when the concentration was above 100 nmol·L-1. Indice of cells arrested in G0/G 1 phases and apoptotic cells increased in a dosage-and time-dependent manner, the difference was significant between Cdk7 ASODN and the sense control (P < 0.01); Characteristic apoptosis in Cdk7 ASODN treated groups were obvious under the transmission electron microscopy. Conclusion: Bioactivities and phosphorylation levels of pRb and Cdk2 decreased after Cdk7 silencing and thus induced obvious G0/G1 phases arrest and apoptosis in HepG2 cell culture in vitro, it is feasible to consider Cdk7 as a novel target for anticancer therapeutics.

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells

The calcium sensing receptor (CaSR) plays an important role for sensing local changes in the extracellular calcium concentration ([Ca2+]o) in bone remodeling. Although the function of CaSR is known, the regulatory mechanism of CaSR remains controversial. We report here the regulatory effect of caveolin on CaSR function as a process of CaSR regulation by using the human osteosarcoma cell line (Saos-2). The intracellular calcium concentration ([Ca2+]i) was increased by an increment of [Ca2+]o. This [Ca2+]i increment was inhibited by the pretreatment with NPS 2390, an antagonist of CaSR. RT-PCR and Western blot analysis of Saos-2 cells revealed the presence of CaSR, caveolin (Cav)-1 and -2 in both mRNA and protein expressions, but there was no expression of Cav-3 mRNA and protein in the cells. In the isolated caveolae-rich membrane fraction from Saos-2 cells, the CaSR, Cav-1 and Cav-2 proteins were localized in same fractions (fraction number 4 and 5). The immuno-precipitation experiment using the respective antibodies showed complex formation between the CaSR and Cav-1, but no complex formation of CaSR and Cav-2. Confocal microscopy also supported the co-localization of CaSR and Cav-1 at the plasma membrane. Functionally, the [Ca2+]o- induced [Ca2+]i increment was attenuated by the introduction of Cav-1 antisense oligodeoxynucleotide (ODN). From these results, in Saos-2 cells, the function of CaSR might be regulated by binding with Cav-1. Considering the decrement of CaSR activity by antisense ODN, Cav-1 up-regulates the function of CaSR under normal physiological conditions, and it may play an important role in the diverse pathophysiological processes of bone remodeling or in the CaSR- related disorders in the body.

Effects of antisense p21 oligodeoxynucleotide on the expression of p21 protein and extracellular matrix of human glomerular mesangial cells in hyperglycemic milieu / 中华肾脏病杂志

Objective To investigate the effects of antisense p21 oligodeoxynucleotide (p21 ASODN) on the expression of p21 protein and extracellular matrix in cultured human glomerular mesangial cells (HGMC) under high glucose medium. Methods HGMCs were transfected with 50 nmol/L or 100 nmol/L p21 ASODN or scrambled control oligodeoxynucleotide (SCODN) using lipofectamine 2000. After incubation under normal (5.5 mmol/L) or high (30 mmol/L) glucose media and different times, HGMCs lysates were collected and the expression of p21, fibronectin and laminin was examined by Western blot. The secretion of fibronectin and laminin by HGMCs in supernatants of culture media was also detected with EOSA. Results High glucose media significantly stimulated the expression of p21, increased the syntheses and secretion of fibronectin and laminin in cultured HGMCs in a time-dependent manner. Transfection of HGMCs with p21 ASODN not only decreased p21 protein level caused by high glucose media, but also attenuated the expression and secretion of fibronectin and laminin in supematants of HGMCs lysates and culture media. Meanwhile, SCODN had no significant effects on the expression of p21, fibronectin and laminin. Conclusions High glucose promotes the expression of p21 , fibronectin and laminin in cultured HGMCs. Transfection of HGMCs with p21 ASODN can decrease p21 protein level caused by high glucose media, and attenuate the expression of fibronectin and laminin in supematants of HGMCs lysates and culture media. Modulation of p21 expression in HGMCs may work as an effective way to mitigate the progression of diabetic nephropathy.

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.