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Objective:To explore the effect of Toll-like receptor 9 (TLR9) signaling pathway activation on the transcriptome in the renal tubular cells.Methods:Mouse primary renal tubular epithelial cells were extracted and cultured. When the degree of cell fusion reached 80%, they were divided into two groups, which were added with 10 μL phosphate buffered saline (PBS, PBS control group) and TLR9 activator cytosine phosphate guanidine oligodeoxynucleotide (CpG-ODN) with a final concentration of 5 μmol/L (CpG-ODN treatment group). The RNA sequencing was performed on the Illumina platform after extraction. DEGseq software was used to analyze the differential expression of genes between the two groups. Goatools and KOBAS online software were used to analyze the differential genes involved signal pathways. Homer software was used to predict transcription factors.Results:Compared with the PBS control group, there were a total of 584 differentially expressed genes in the CpG-ODN treatment group, of which 102 were up-regulated and 482 were down-regulated. The most significantly enriched gene ontology (GO) terms of differentially expressed genes included response to interferon-β, defense response to virus and other inflammatory pathway. The most significantly enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathways included 2'-5'-oligoadenylate synthase activity, regulation of ribonuclease activity, negative regulation of virus life cycle, cellular response to interferon-βand defense response to protozoan. The results of transcription factor prediction showed that interferon regulatory factor 3 (IRF3) was the most significantly enriched transcription factor in the promoter sequence of differential genes; the most significant transcription factor downstream of TLR9 was IRF3, and other predicted transcription factors such as transcription factor 21 (TCF21), zinc finger protein 135 (ZNF135), and PR domain containing 4 (PRDM4) might be new candidates for TLR9 signaling pathway.Conclusion:CpG-ODN activates TLR9 signaling pathway, and primary renal tubular epithelial cells can directly respond to CpG-ODN stimulation and undergo transcriptome changes, which provides a basis for further research on the molecular mechanism of TLR9 pathway in sepsis induced acute kidney injury.
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The potential application of dendritic cells (DC) sensitized with cytosine-phosphoric acid-guanine (CpG) oligodeoxynucleotide (ODN) and tumor antigen as a vaccine against murine melanoma was investigated with freshly isolated mouse bone marrow-derived dendritic cells. For the DC vaccine preparation, DC were sensitized with the B16 tumor antigen and CpG ODN was used to promote further maturation of the DC. The immunogenic activity of the vaccine was evaluated in vitro by determining the proliferation of T lymphocytes and the killing effect of cytotoxic T lymphocytes (CTL) on B16 tumor cells. The DC vaccine was injected intraperitoneally and tumor inhibition in mice bearing B16 xenografts was examined. All mice were cared for under an approved SIMM Institutional Animal Care and Use Committee (IACUC) protocol. In vitro, this DC vaccine promoted the proliferation of T lymphocytes and showed a potent killing effect on the target B16 cells. In vivo experiments showed that after treatment or pre-immunization both the tumor volume and weight were significantly decreased. The DC vaccine with CpG ODN and tumor antigen exhibited an inhibitory effect against melanoma, providing a potential method for melanoma cancer treatment.
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Objective@#To investigate the curative effect of local application of CpG-oligodeoxynucleotide (CpG-ODN) combined with 4-1BB monoclonal antibody in hepatoma-bearing mice, and to evaluate the effect of 4-1BB monoclonal antibody on CpG-ODN immunotherapy.@*Methods@#H22 single cell suspension was injected subcutaneously into the axilla and four limbs of the BALB/c male mice to establish a tumor-bearing mice model. After 7 days, 30 mice with corresponding tumor-bearing volume were screened and randomly divided into model control group, CpG group and CpG+4-1BB group, and the drug was injected into the tumors of left lower extremity. The same batch of normal mice was selected as normal control group. Survival of mice was recorded. Tumor-bearing volume and organ index were calculated. Serum levels of interleukin (IL) - 12 and interferon (IFN) gamma and spleen CD8+T lymphocyte ratio were measured. The measurement data were analyzed by analysis of variance. The survival rate of each group of mice was analyzed by log-rank test.@*Results@#Mice in the model control group with tumor-bearing volume had a sustained growth before the execution. CpG group and the CpG+4-1BB group [(976.08 ± 29.55) mm3, (47.25 ± 0.93) mm3)] tumor-bearing volume was decreased than model group [(1 336.52 ± 39.40) mm3] (F = 5 329.273, P < 0.05). CpG+4-1BB group distant tumor-bearing volume [(611.83 ± 113.02) mm3] was decreased than model group and CpG group [(1 406.62 ± 51.09) mm3, (1 380.01 ± 51.44) mm3] (F = 247.160, P < 0.05), but there was no significant difference between the CpG group and the model group (P > 0.05). Serum IL-12 concentration (23.90 ± 2.33 pg/ml), IFN-γ concentration (103.02 ± 6.10 pg/ml) and spleen CD8+T cell ratio (4.54 ± 0.62%) in the model group were lower than those in the normal group (P < 0.05). Serum IL-12 concentration in CpG group and CpG+4-1BB group (29.21 ± 2.23 pg/ml, 37.04 ± 1.49 pg/ml), IFN-γ concentration (116.12 ± 4.08 pg/ml, 138.65 ± 1.72 pg/ml), CD8+T cell ratio (6.65 ± 0.64%, 12.73 ± 0.88%) were higher than the model group, while CpG+4-1BB group was higher than the CpG group (P < 0.05). The survival rate of CpG+4-1BB group was higher than that of model group and CpG group (χ2 = 25.544, P < 0.05), but there was no significant difference between CpG group and model group (P > 0.05). There was no significant difference in organ index between the four groups (P > 0.05).@*Conclusion@#4-1BB monoclonal antibody combined with CpG-ODN therapy can shrink hepatoma-bearing capacity, inhibit the growth of distant tumors and significantly prolong the survival time of mice.
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Objective: To investigate the effects of oligodeoxynucleotide (ODN) YW002 on the proliferation, cell cycle, apoptosis and early osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs), and to clarify the regulation effect of ODN YW002 on the biological properties of hPDLSCs. Methods: The HPDLSCs were cultured until the third to the fifth generations. The cells were divided into PBS blank control group, ODN MT01 group and ODN YW002 group and incubated for 1, 2, 3 and 5 d. The proliferation activities of the hPDLSCs in various groups were detected by MTT assay. The cell cycle and apoptosis of the hPDLSCs in various groups were detected by flow cytomety. The alkaline phosphates (ALP) activities of the hPDLSCs in various groups were detected by alkaline phosphatase colorimetric assay. Results: Compared with blank control group, the proliferation activities of hPDLSCs in ODN YW002 group at 1 and 3 d after culture were increased (P 0 . 05). Compared with ODN MT01 group, the proliferation activity of hPDLSCs in ODN YW002 group at 1 d after culture was decreased (P 0 . 05). Compared with blank control and ODN MT01 group, the percentage of hPDLSCs in Go/Gi phase in ODN YW002 group was decreased at 1 d after cultrue, but there was no significant difference (P > 0 . 05); the percentage of hPDLSCs in S phase was increased at 1 d after culture, but there was no significant difference (P> 0. 05). Compared with blank control group, the early and late apoptotic rates of hPDLSCs in ODN YW002 group were decreased at 1 d after culture (P 0 . 05). Compared with ODN MT01 group, the early and late apoptotic rates of hPDLSCs in ODN YW002 group were decreased at 2 d after culture (P < 0 . 05). Compared with blank control group, the ALP activities of hPDLSCs in ODN YW002 group were increased at 1, 3 and 5 d after culture (P < 0 . 01). Compared with ODN MT01 group, the ALP activities of hPDLSCs in ODN YW002 group were increased at 1 and 5 d after culture (P < 0 . 05), but the ALP activity was decreased at 3 d after culture (P < 0.05). Conclusion: ODN YW002 can promote the proliferation of hPDLSCs by inhibiting the apoptosis, and increase the ALP activity, suggesting that ODN YW002 has the function of promoting the osteogenic differentiation of hPDLSCs.
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Objective:To investigate the partial delivery ability in vivo of oligonucleotide MT01 mediated by polyethyleneimine(PEI) derivative PEN, and to illuminate its effect on the alveolar bone reconstruction and the biological safety in the rats with experimental tooth movement. Methods:Forty-eight Wistar male rats were randomly divided into PEN,MT01, sulfuration-modified MT01 (MT01s), and PEN/MT01 groups. The rat experimental tooth model was established, and the above four drugs were injected into the mesial buccal mucosa of the first molar in the left sides of the rats as drug intervention sides,and the PBS were injected into the mesial buccal mucosa of the first molar in the right sides as PBS control sides;once every 3 d. After 14 d, the rats were sacrificed. HE staining was used to detect the histopathological changes of important organs of the rats;the photos and X-ray films were applied to measure the distance of tooth movement, and RT-PCR was used to detect the expression levels of the osteogenic marker genes Runt-related transcription factor 2(Runx2),special protein 7(SP7) and osteocalcin(OCN) mRNA. Results:After local injection of the above drugs, no obvious inflammatory cell infiltration and structural differences were found in the main organs of the rats and no obvious abnormalities were found in all organs tissues. Compared with the PBS control sides, the movement distance of the first molars in the drug intervention sides in MT01, MT01s, and PEN/MT01 groups was reduced (P0.05). The data of the distance between the two teeth of the rats in various groups was PEN/MT01 group > MT01s group > MT01 group>PEN group, and the difference between PEN/MT01 group and MT01s group was statistically significant (P0.05). Conclusion:PEI derivative PEN can transfer MT01 into the cell safely and efficiently; it can increase the expression levels of Runx2, SP7 and OCN mRNA in the periodontium tissue and decrease the tooth movement distance in the experimental tooth movement rats.
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@#Objective: To investigate the role of CpG ODN (CpG oligodeoxynucleotide) adjuvant in enhancing the anti-bladder cancer response induced by MAGE-3 (melanoma antigen gene -3) antigen and its molecular mechanism. Methods: Mononuclear cells were isolated from HLA-A2 type peripheral blood of healthy donors by Ficoll method to prepare mature DC by conventional means. DC surface markers were detected by flow cytometry. MTT assay was used to detect the promotion effect of DCs sensitized by different means (MAGE-3, CpGODN, MAGE-3+CpG ODN, irrelevant control antigen) on the proliferation of T lymphocytes and the killing effect of CTL on BIU-87 tumor cells. The tumor mass of nude mice bearing BIU-87 bladder cell xenograft were examined on Day 7 and 11 after CpG ODN+MAGE-3 sensitized DC treatment. The expression of Bcl-2/Bax protein was detected by Western blotting while the proliferation level of xenograft cells was detected by MTT assay. Results: DCs sensitized by CpG ODN combined with MAGE-3 antigenic peptides could promote the proliferation of T lymphocytes and significantly enhance the killing effect of CTLon target BIU-87 cells (P< 0.05). Compared with other sensitized DCs, in vivo experiments showed that 7 and 11 days after treatment, both the tumor volume and weight were significantly reduced (all P<0.05), and the proliferation ability of xenograft tumor was decreased (P<0.05). Compared with other sensitization means, CpG ODN+MAGE-3 especially exhibited obvious inhibitive effect on tumor growth on Day 11, and significantly promoted the proliferation of splenic monocytes of tumor bearing mice (P<0.01); moreover, Bcl-2 expression in xenograft tissues significantly decreased（P<0.01）while Bax expression significantly increased（P<0.05 or P<0.01）on Day 3 after treatment. Conclusion: CpG ODN can promote the inhibitory effect of MAGE-3 sensitized DC on bladder cancer BIU-87 cells, which will provide experimental basis for clinical application of DC vaccine in bladder cancer treatment.
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Objective:To investigate the effect of MT01 on the differentiation of osteoblasts under infected condition through determining the expression level of collagen Ⅰ (Col Ⅰ )mRNA in MG63 cells treated with Porphyromonas gingivalis (Pg).Methods:The cultured MG63 cells were divided into blank control,MT01,Pg, and MT01+Pg groups.MT01 at a concentration of 1 mg·L-1 was added into the MG63 cells,and the cells were incubated for 3 h.The cells treated with PBS (1 mg·L-1 )were used as control group.Then Pg (MOI=100∶1) was added.Real-time PCR was used to detect the expression levels of ColⅠ mRNA in MG63 cells at 2,4,6,8, 12 and 24 h after incubation.Results:Compared with blank control group,the levles of ColⅠ mRNA in the MG63 cells in MT01 and MT01 + Pg groups had no significant changes at 2 and 4 h (P > 0.05);the Col Ⅰ mRNA expression levels in MT01 group at 8,12 and 24 h were increased (P < 0.05 or P < 0.01).Compared with Pg group,the expression levels of ColⅠ mRNA in MT01+ Pg at 2 and 4 h were decreased,but the expression levels of ColⅠ mRNA were increased at 6,8,12 and 24 h (P <0.05 or P <0.01).Conclusion:MT01 can up-regulate the expression level of Col Ⅰ mRNA in the infected MG63 cells; MT01 could promot the differentiation of osteoblasts under infected condition.
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Objective To evaluate the influence of preconditioning with and anti-myosinmonoclonal antibody (mAb2G4)-nuclear factor-kappa B decoy oligodeoxynucleotide (ODN)-lipofectamine (lip) on hypoxia-reoxygenation (H/R) injury in H9c2 cardiomyocytes.Methods H9c2 cardiomyocytes were seeded in 6-well plate at the density of 1×105/ml (2 ml/well),and were divided into 3 groups (n=9 each) using a random number table:control group (group C),H/R group and mAb2G4-ODN-lip group (group MOL).The cells underwent 2 h of hypoxia in an air-tight bag,followed by 1 h reoxygenation.In MOL group,the cells were treated with mAb2G4-ODN-lip (2 μg ODN) for 4 h and then cultured in the common culture medium for 8 h before hypoxia.At the end of reoxygenation,proliferation of cells was measured using MTT assay,and the cells and supernatant of the culture medium were collected to determine the activity of lactate dehydrogenas (LDH),content of malondialdehyde (MDA),concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) (by ELISA).The rate of proliferation inhibition was calculated.Results Compared with group C,the rate of proliferation inhibition,LDH activity,MDA content,and concentrations of TNF-α and IL-6 were significantly increased in the other two groups.Compared with group H/R,the rate of proliferation inhibition,LDH activity,MDA content,and concentrations of TNF-α and IL-6 were significantly decreased in MOL group.Conclusion mAb2G4-ODN-lip can mitigate H/R injury in H9c2 cardiomyocytes.
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Objective To study the treatment effects of newborn mice murine Cytomegalovirus (MCMV) hepatitis by using CpG Oligodeoxynucleotide 2395 (CpG ODN2395).Methods Specific pathogen-free BALB/c newborn mice were divided into 3 groups according to the broods randomly:control group,virus group and treatment group.In control group 9 g/L sodium chloride 20 × 10-6 L was intraperitoneally injected every other day.In virus group MCMV (TCID50 =108.31/L) 20 × 10-6 L was intraperitoneally injected once and 9 g/L sodium chloride solution 20 × 10-6 L was intraperitoneally injected every other day.In treatment group murine MCMV(TCID50 =10S31/L) 20 × 10-6 L was intraperitoneally injected once and from the first day CpG ODN2395 20 mg/kg was intraperitoneally injected every other day.Growths and development of mice were observed.Murine body weights were measured.Pathology of livers was observed by means of hematoxylin and eosin stain.The levels of serum ALT and IFN-α were measured by adopting enzyme linked immunosorbent assay.MCMV loads in liver were measured by way of polymerase chain reaction.The expression levels of IFN-α mRNA in liver were detected by using reverse transcription polymerase chain reaction.The expression levels of Toll-like receptor 9 (TLR9) and myeloid cell differentiation factor 88 (MyD88) in liver were detected by adopting Western blot.The results were analyzed by using SPSS 16.0 statistics software.Results 1.Compared with control group and treatment group,growth and development of virus group mice fell behind and on day 7,14 body weights were lowest(F =18.919,25.543,all P ＜ 0.05).Growth and development of treatment group mice were between control group and virus group.Body weights of treatment group mice were lower than those of control group,and there was statistical difference on day 7 (t =3.187,P ＜ 0.05).2.Compared with control group and treatment group,the levels of ALT in virus group was highest.ALT levels of treatment group were higher than those of control group and treatment group(F =11.407,11.791,154.656,all P ＜ 0.05).3.The pathologic change of liver tissue:the HAI of virus group reached the peak on day 3,then decreased gradually.The HAI of treatment group also reached the peak on day 3,but liver damage was obviously less than those of virus group.The liver damage also relieved gradually and the mean of HAI was obviously lower than that of virus group on day 7 and 14.4.MCMV DNA in liver was negative at control group.The magnitude of viral loading in livers of virus group was higher than that of treatment group.5.The levels of IFN-α in treatment group and virus group reached a peak on day 3 and declined gradually on day 7,14.The levels of IFN-α on treatment group were higher than that of virus group and control group.The levels of IFN-α virus group were higher than those of control group,but had no statistical difference.6.The mRNA expression of IFN-α in livers of treatment group and virus group began to increase on day 3 and reached a peak on day 7,and declined on day 14.The mRNA expression of IFN-α was higher than that of virus group and control group.The mRNA expression of virus group was higher than that of control group.7.The expressions of TLR9 and MyD88 in livers of treatment group were higher than that of virus group and control group.The expressions of TLR9 and MyD88 of virus group were higher than those of control group.Conclusions CpG ODN2395 can obviously improved liver function and histopathological lesions and reduce MCMV DNA load within liver tissues as well.CpG ODN2395 can improve the expression levels of TLR9 in liver and activate secretion interferon alpha by MyD88-dependent pathway,which were likely to play an important role in its treatment of murine CMV infection.
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Objective To observe the distribution of oligodexynucleotide (ODN)MT01 in main organ tissues of the rats at different time points and to discuss the regularity of the distribution of MT01 preliminarily. Methods 60 male Wistar rats were randomly divided into experimental group(n=30)and control group(n=30). The rats in experimental group was locally injected with Cy5 labeled MT01 in gingival mucosa,whereas the rats in control group were injected with MTO1.The samples of rat lung,liver spleen,kidney,heart,and brain tissues were collected at 15 min,1 h,4 h,8 h,16 h,1 d,2 d,3 d,4 d,and 5 d after injection,and the distribution of MT01 fluorescence was observed by laser scanning confocal microscope.The ratio of fluorescence positive cells indicated the amount of MT01 that had been taken up by different organs.Results No positive fluorescence cells were observed in control group.Whereas,in experimental group ,the positive fluorescence cells were detected in the tissue samples of lung,liver,spleen and kidney but not in the tissue samples of heart and brain.The positive fluorescence cells distributed focally in kidney tissue and presented primarily in the cytoplasm of renal tubular epithelial cells.The ratios of positive fluorescence cells changed regularly with time in liver, spleen and kidney tissues and the highest level was detected at 4,3 and 4 d after injection.No distinct regularity of the ratio of positive fluorescence cells was observed in lung tissue.Conclusion MT01 can be taken up by liver,spleen and lung tissue and primarily by kidney with regularity in distribution.
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Objective To prepare and evaluate flutide-loaded PLGA nanoparticles modified with cell-penetrating peptide-TAT.Methods The sequence of TAT was synthesized with florenl methyoxycarbonyl amino acids .The purity and molecular weight of TAT were determined using RP-HPLC and MALDI-TOF-MS.PLGA was modified with the TAT peptide and then prepared into flutide-loaded nanoparticles ( TAT-PLGA NPs) with the double emulsion method .The physical and chemical properties were evaluated , including size distribution, Zeta potential, SEM of nanoparticles , loading ratio of drug content and release profiles of TAT-PLGA NPs in vitro.The cytotoxicity of TAT-PLGA NPs was evaluated by CCK-8 methods.Results The purity of synthesized TAT was 95.6%, and molecular weight was 1495.8.The mean diameter,Zeta potential, drug loading ratio of TAT-PLGA nanoparticals were (159.5 ±2.1) nm, -(1.87 ±0.6) mV, and (5.75 ±0.17)μg/mg, respectively.The nanoparticles observed by transmission electron microscopy (TEM) had a spherical shape and uniform size without aggregation .In vitro release test showed sustained release of flutide from TAT-PLGA nanoparticles .Cell proliferation assay revealed that the TAT-PLGA nanoparticles did not damage the cell growth in vitro and showed good compatibility.Conclusion TAT-PLGA nanoparticles are prepared successfully by double emulsion method,and have sustained-release effect and good compatibility in vitro.They have potential application prospect in prevention and treatment of influenza .
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Objective To investigate the effect of S-phase kinase-associated protein 2 antisense oligodeoxynucleotide (Skp2 ASODN) on the growth and proliferation of colorectal cancer cells SW480 and its mechanism .Methods The liposome-mediated dif-ferent concentrations of Skp2 ASODN was adopted to transfect SW480 cells ,the final concentrations of 0 .050 ,0 .125 ,0 .250 ,0 .500 , 1 .000 ,2 .000 ,4 .000 μmol/L were respectively set up with Skp2 nonsense oligodeoxynucleotides(NSODN) and blank group as con-trol .Then the inhibited effect of growth and proliferation of colorectal cancer cells were measured by the inverted microscope obser-vationandmethylthiazolyltetrazolium(MTT),thecellcyclewassurveyedbytheflowcytometry(FCM).TheexpressionofmRNA and protein of Skp2 and P27kip1 were inspected by reverse transcription-polymerase chain reaction(RT-PCR) and immunocytochem-istry methods .Results The inverted microscope observed that the SW480 cells had no change in form and grew at a slower speed . When the Skp2 ASODN concentration reached 0 .125 μmol/L ,the inhibition rate was 14 .48% (P0 .05) ,but its protein expression was elevates obviously (P<0 .01) .Conclusion Skp2 ASODN can inhibit the growth and proliferation of SW480 cells .Its possible mechanism is that the ubiq-uitin degradation of the Skp2 to P27kip1 is decreased by the gene occlusion effect of Skp2 ASODN ,thus the cell cycle progression is arrested .
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Objective To investigate the expression of matrix metalloproteinase-2 (MMP-2)and matrix metalloproteinase-9(MMP-9) in MIA PaCa-2 cells blocked by AS-ODN cultured in hypoxia.Methods Heparanase(Hpa) expression of MIA PaCa-2 cells was blocked by AS-ODN and cultured in hypoxia.The expression of MMP-2 and MMP-9 mRNA and proteins in cell lysate was evaluated by RT-PCR and Western blot respectively,and the enzymatic activities of MMP-2 and MMP-9 in supernatants were detected by gelatinase activity assay.Results Hypoxia stimulated mRNA and protein expression of MMP-9 in cultured MIA PaCa-2 cells and elevated at 6h,12 h(P ＜0.05)and 24 h(P ＜ 0.01).When Hpa expression was inhibited by AS-ODN,the expression of MMP-9 mRNA and protein as well as the gelatinase activity in supernatant decreased dramatically at 12 h and 24 h,especially at 24h(P ＜0.01),however,no significant difference of MMP-2 expression and gelatinase activity was observed after AS-ODN transfection.(P ＞ 0.05).Conclusion In hypoxia,MMP-9 expression,either mRNA or protein in cultured MIA PaCa-2 cells,increased gradually accompanied with elevated gelatinase activities.When the heparanase expression was inhibited,the MMP-9 mRNA and protein,as well as the gelatinase B activity in supernatant,were decreased dramatically at 12h and 24h,however,no significantly differences of MMP-2 expression and gelatinase A activity were observed after the AS-ODN transfection.
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Objective To explore the combination effect of unmethylated cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN) 1826 and X-rays on Lewis lung cancer in mouse and the dose response of CpG ODN.Methods The tumor-bearing mouse model was established by injecting Lewis lung cancer cells into the right infra-axillary dermis of mouse.Sixty-four C57BL /6 J mice were evenly randomized into eight groups with 8 mice each:control group,IR group,CpG OND1826 0.15 mg group,CpG OND1826 0.3 mg group,CpG OND1826 0.45 mg group,CpG OND1826 0.15 mg + IR group,CpG OND1826 0.30 mg+ IR group,and CpG OND1826 0.45 mg + IR group.On the 1st,2nd,and 9th days,CpG ODN was injected into mouse.After 3 hours of injection,the mice were start to irradiate with X-rays once a day on the 2nd-6th days,and the total dose was 12.50 Gy.Tumor growth and TGD were measured,and the apoptosis of tumor cells were examined with TUNEL.Results The Lewis lung cancer-bearing model was successfully established in all mice.Under the treatments of CpG OND1826 and irradiation,the tumor volumes were smaller than that of control group,and the tumor volumes of CpG OND1826 0.45 mg+IR group was the smallest.TUNEL results revealed that the apoptosis rate were (2.40 ± 0.51 )％ in control group,(5.62 ±0.50)％ in IR,(7.13±0.83)％ in CpG OND1826 0.15 mg,(11.63±1.06)％ in CpG OND1826 0.3 mg,(19.13 ±0.83)％ in CpG OND1826 0.45 rag,( 12.88±0.83)％ in CpG OND1826 0.15 mg+ IR,(20.57±2.37)％ in CpG OND1826 0.3 mg+ IR,and (28.17 ±3.31)％ in CpG OND1826 0.45 mg + IR group,and thus the apoptosis rate of every therapy group was higher than that in control ( t=11.15,7.91,17.82,39.48,24.73,16.61 and 17.05,P＜0.05).The apoptosis rates of CpG ODN1826 plus X-ray irradiation group were significantly higher than those in IR alone ( t =13.78,15.08 and 17.47,P＜0.05 ) or CpG ODN group (t=18.53,9.66and7.51,P＜0.05).Conclusions CpG ODN1826 can dramatically increase the efficiency of radiotherapy by inhibiting tumor growth and promoting lumor apoptosis.
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ObjectiveTo investigate the effect of Smad7 antisense oligodeoxynucleotide (ASODN) on proliferation in human pancreatic cancer cell line SW1990,with a focus on the expression of matrix metalloproteinase-2(MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2).To explore the underlying mechanism of the role of Smad7 in the pathogenesis and development of pancreatic cancer.MethodsSmad7 ASODN was transfected into SW1990 cells through lipofectamine.Nosense oligodeoxynucleotide (NSODN),ASODN and lipofectamine was used as control. The transfection efficiency was assessed by fluorescence microscopy and flow cytometry.The expressions of Smad7,MMP-2 and TIMP-2 in transfected cells were detectedby RT-PCR and Western blot.Cell viability was assessed by dimethyl thiazoldiphenyltetrazoliumbromide (MTT) method. Results Smad7 was expressed in SW1990 cells.The transfection efficiency of SW1990 was 81.2％.The expressions of Smad7 mRNA were 0.34 ± 0.06,0.95 ±0.07,1.03 ± 0.11 in transfected group,ASODN and lipofectamine group; and the expressions of MMP-2 mRNAwere 0.54 ± 0.08,1.15 ± 0.13,1.27 ± 0.16 ; and the expressions of TIMP - 2 mRNA were 0.26 ±0.07,0.72 ± 0.13,0.78 ± 0.17,the mRNA expressions were significantly reduced in Smad7 ASODN transfected group,compared with other two groups (P ＜0.01 ).The expressions of Smad7 protein were 0.14 ± 0.03,0.29 ± 0.05,0.28 ± 0.07 in transfected group,ASODN and lipofectamine group; the expressions of MMP-2 protein were 0.17 ±0.02,0.29 ±0.05,0.31 ±0.04,and the expressions of TIMP-2 protein were 0.20 ± 0.03,0.41 ± 0.11,0.43 ± 0.09,the protein expressions were significantly reduced in Smad7 ASODN transfected group,compared with other two groups (P ＜0.01 ).The A490 values of proliferation were 0.83 ± 0.03,1.02 ±0.02,0.99 ±0.02 in transfected group,ASODN and lipofectamine group,the proliferation were significantly reduced in Smad7 ASODN transfected group,compared with other two groups (P ＜0.01 ).ConclusionsSmad7 ASODN could effectively inhibit the expressions of Smad7,therefore decrease the expressions of MMP-2,TIMP-2 and reduce the proliferation.
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Objective To investigate the effects of intrathecally cyclic AMP response element-binding protein(CREB) antisense oligodeoxynucleotide (ODN) on neuropathic pain behaviors.Methods Using mouse model of neuropathic pain induced by chronic constriction injury of sciatic nerve (CCI),24 male C57BL/6 mice successfully received intrathecal catheter implantation and without motor dysfunction were randomly divided into 4groups(n=6):Saline group(NS),CREB sense ODN group(S),CREB missense ODN group(M),CREB antisense ODN group(A).Mice in NS,S,M and A were intrathecally treated with Saline 5μ l,Sense ODN 5μg/5μl,Missense ODN 5μg/5μl and Antisense ODN 5μg/Sμl once daily on day 1 ～6 after CCI respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency(PWTL) were tested on day 1 before CCI and day 1,3,5,7,10,14,17,21 after CC(I).Results Mice in A group maintained the pain thresholds in the baseline and lasted at least 7 days after CCI ( 7 d,PWMT:( 0.81 ± 0.20 ) g vs ( 1.00 ± 0.19 ) g,P ＞ 0.05 ;PWTL:(5.96 ± 0.69) s vs (6.93 ± 1.08 ) s,P ＞ 0.05 ).The withdrawal thresholds in the ipsilateral hind paws of the mouse were significantly lower than baseline in A group on day 10 after CCI( 10 d,PWMT:(0.56 ±0.19)g vs (1.00±0.19)g,P＜0.05; PWTL:(3.93 ±0.28)s vs (6.93 ± 1.08)s,P＜0.05).Compared with NS group ( 10 d,PWMT:(0.56 ±0.19)g vs (0.37 ±0.08)g,P＜0.05; PWTL:(3.93 ±0.28)s vs (3.14 ±0.45)s,P＜0.05),S group,M group,the withdrawal thresholds of A group was significantly elevated on day 10 after CCI.These effects lasted up to at least day 21 after CCI.Conclusion Intrathecally treated with CREB antisense ODN in the development of neuropathic pain induced by CCI completely improved pain behaviors during the course of injection,and the effects of relief pain lasted at least 15d after no injection.
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Objective To select an efficient method to inhibit telomerase activity, antisenseoligodeoxynucleotide and plasmid-vector mediated RNAi against hTERT were used to inhibit telomerase activity. The inhibiting effects of the two methods were compared. Methods Against hTERT mRNA, siRNA and oligodeoxynucleotide were designed and transfected into K562 cells by liposome. Effective and specific siRNA strands were selected and then plasmid was constructed and transfected into K562 cells; followed by analysis of the results. Results hTERT mRNA were detected after the three chemo-synthesized strands were transfected. It was found that si-hTERT-1 and si-hTERT-2 were effective, but si-hTERT-3 had no effect. The inhibiting effect of hTERT mRNA lasted only 48 h and disappeared at 72 h. Two siRNA strands were sieved and plasmids were constructed and transfected into K562 cells. In the P-1 group, hTERT mRNA was 0.39±0.13 at 48 h, 0.57±0.32 at 72 h. In the P-2 group, hTERTmRNA was 0.55±0.20 at 48 h, 0.88±0.23 at 72 h.In the P-1 group, the relative telomerase activity was 0.42±0.07 at 48 h, 0.31±0.08 at 72 h. In the P-2 group was 0.49±0.27 at 48 h, 0.39±0.03 at 72 h. The best concentration of siRNA was 100 μmol/L. The best concentration of ASODN was 0.6 μ mol/L. hTERTmRNA was 0.42±0.16 at 24 h, 0.71±0.18 at 48 h. Relative telomerase activity was 0.52±0.002 at 24 h, 0.482±0.018 at 48 h. Conclusion Both ASODN and RNAi targeting hTERT can inhibit the expression of hTERT mRNA, and then inhibit telomerase activity. The inhibiting effect is closely relative to the targeting site. The inhibiting effect of RNAi is better than that of ASODN. RNAi has better efficiency and lasts for a longer time. Plasmid mediated RNAi has better inhibiting effect than the chemo-synthesized siRNA.
ABSTRACT
CpG oligodeoxynucleotides (ODN) have potent immunostimulatory effects and can enhance the anti-cancer activity of cancer treatments. CpG ODN directly induced the activation and maturation of plasmacytoid dendritic cells, stimulated the secretion of Th1-type cytokines, and enhanced the differentiation of B cells into antibody-secreting plasma cells. CpG oligodeoxynucleotides as vaccine adjuvants can enhance both the humoral and cellular responses to antigens in some clinical trails. CpG ODN was applied in several clinical trials as an adjuvant of tumor vaccine. CpG ODN alone had anti-tumor activity and had synergistic effects with other anti-tumor therapies including monoclonal antibodies, chemotherapy, radiotherapy, cytokines, etc. Compared with standard regimen, in the phase Ⅲ clinical trial CpG ODN did not prolong the median survival time and induced severe adverse reaction in the treatment of ⅢB-Ⅳ stage non-small cell lung cancer. But CpG ODN had definite anti-cancer activity in other clinical trials. The safety and efficacy of CpG ODN in anti-tumor therapy needs to be further verified in clinic.
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Objective:To study the mechanism of apoptosis in laryngeal carcinoma cell induced by Stat3 antisense oligodeoxynucleotide (ASODN).Method:The designed Stat3 ASODN was transferred into laryngeal cacinoma Hep-2 cell by lipofection. Expression of Bcl-2, Bax and C-Myc were detected by Western blot and PCR.Result:Western blot and PCR results demonstrated that Stat3 ASODN could significantly increased the expression of Bax and decreased the expression of Bcl-2 and C-Myc when the concentration of antisense oligodeoxynucleotide were heightened.Conclusion:Stat3 ASODN participate in apoptosis by enhancing the expression of Bax and reducing the expression of Bcl-2 and C-Myc.
ABSTRACT
Excessive extracellular matrix (ECM) accumulation is the main feature of chronic renal disease including diabetic nephropathy. Plasminogen activator inhibitor (PAI)-1 is known to play an important role in renal ECM accumulation in part through suppression of plasmin generation and matrix metalloproteinase (MMP) activation. The present study examined the effect of PAI-1 antisense oligodeoxynucleotide (ODN) on fibronectin upregulation and plasmin/MMP suppression in primary mesangial cells cultured under high glucose (HG) or transforming growth factor (TGF)-beta1, major mediators of diabetic renal ECM accumulation. Growth arrested and synchronized rat primary mesangial cells were transfected with 1 microM phosphorothioate-modified antisense or control mis-match ODN for 24 hours with cationic liposome and then stimulated with 30 mM D-glucose or 2 ng/ml TGF-beta1. PAI-1 or fibronectin protein was measured by Western blot analysis. Plasmin activity was determined using a synthetic fluorometric plasmin substrate and MMP-2 activity analyzed using zymography. HG and TGF-beta1 significantly increased PAI-1 and fibronectin protein expression as well as decreased plasmin and MMP-2 activity. Transient transfection of mesangial cells with PAI-1 antisense ODN, but not mis-match ODN, effectively reversed basal as well as HG- and TGF-beta1-induced suppression of plasmin and MMP-2 activity. Both basal and upregulated fibronectin secretion were also inhibited by PAI-1 antisense ODN. These data confirm that PAI-1 plays an important role in ECM accumulation in diabetic mesangium through suppression of protease activity and suggest that PAI-1 antisense ODN would be an effective therapeutic strategy for prevention of renal fibrosis including diabetic nephropathy.