Expression of decoy receptor 3 and its signaling pathway in ankylosing spondylitis and its clinical significance / 中华风湿病学杂志

Objective:To investigate the expression and clinical significance of decoy receptor 3 (DcR3) and its signal pathway-related molecules in PBMCs of patients with ankylosing spondylitis (AS).Methods:Peripheral blood samples, clinical data and laboratory test results were collected from 100 patients with ankylosing spondylitis [50 patients with AS activity (ASA), 50 patients with AS stability (ASS)], 30 patients with osteoarthritis and 30 patients with gouty arthritis (as disease control group), and 60 healthy controls (HC). The mRNA expression levels of DcR3 and its signal pathway related genes (DR3, TL1A, Fas, FasL, LIGHT, LIGHTR, LTβR) were measured by real-time fluorescence quantitative polymerase chain reaction. Measurement data among the three groups in normal distribution were analyzed by t test or one-way analysis of variance, pairwise comparisons using LSD- t test, non-normal distribution data were analyzed by Mann-Whitney test or Kruskal-Wallis H test, χ2 test was used for correlation analysis of categorical variables. Correlation analysis between variables were analyzed using Spearman correlation analysis. Results:① By comparing the AS group, disease control group and HC group, the expression levels of DcR3 mRNA and DR3 mRNA in the AS group were lower than those in disease control group and HC group, and DcR3 mRNA and DR3 mRNA in disease control group were lower than those in the HC group {DcR3mRNA: [6.21 (3.89, 10.70)]×10 -4vs [9.51 (5.89, 16.65)]×10 -4vs [17.81 (11.27, 24.20)]×10 -4, H=55.28, P<0.001; DR3 mRNA: [41.05 (24.09, 66.95)]×10 -4vs [58.28 (28.41, 94.38)]×10 -4vs [94.79 (54.07, 144.51)]×10 -4, H=37.10, P<0.001}. The expression level of TL1A mRNA in the AS group was higher than that in disease control group {[14.71(4.91, 42.22)]×10 -4vs [4.00(1.07, 16.60)]×10 -4vs [7.70 (3.52, 27.83)]×10 -4, H=17.71, P<0.001}; The expression level of Fas mRNA in AS group and disease control group was lower than that in HC group {[20.99(4.63, 62.89)]×10 -4vs [23.97(15.82, 38.99)]×10 -4vs [78.45 (27.32, 146.46)]×10 -4, H=31.17, P<0.001}. The expression level of FasL mRNA in AS group was higher than that in disease control group and HC group {[42.87(6.57, 91.21)]×10 -4vs [5.45(2.83, 10.32)]×10 -4vs [6.88 (4.57, 23.79)]×10 -4, H=46.42, P<0.001}. The expression level of LIGHTR mRNA in AS group was lower than that in disease control group {[52.66 (7.20, 143.21)]×10 -4vs [98.80 (53.11, 166.24)]×10 -4vs [63.47(40.85, 138.07)]×10 -4, H=11.96, P<0.001}. There were no significant differences in LIGHT mRNA and LTβR mRNA among all groups ( H=0.86, P>0.05; H=3.18, P>0.05). ②The expression levels of DcR3 mRNA, DR3 mRNA and Fas mRNA in ASA group and ASS group were lower than those in HC group. DcR3 mRNA in ASA group was higher than that in ASS group, and DR3 mRNA in ASA group was lower than that in ASS group {DcR3 mRNA: [7.28 (4.92, 16.56)]×10 -4vs [4.59 (2.49, 7.03)]×10 -4vs [17.81 (11.27, 24.20)]×10 -4, H=62.63, P<0.001; DR3 mRNA: [30.93(16.18, 66.66)]×10 -4vs [47.17(29.91, 67.40)]×10 -4vs [94.79(54.07, 144.51)]×10 -4, H=41.48, P<0.001; Fas mRNA: [20.04(3.29, 62.30)]×10 -4vs [22.49(5.63, 64.79)]×10 -4vs [78.45(27.32, 146.46)]×10 -4, H=23.54, P<0.001}. The expression levels of TL1A mRNA and LTβR mRNA in the ASA group were higher than those in the ASS group and the HC group {TL1A mRNA: [32.36(10.09, 97.84)]×10 -4vs [9.98(1.29, 21.63)]×10 -4vs [7.70(3.52,27.83)]×10 -4, H=21.14, P<0.001; LTβR mRNA: [6.13(2.16,20.06)×10 -4vs [2.13(0.53,8.04)]×10 -4vs [2.72 (1.24,5.73)]×10 -4, H=12.86, P<0.001}. The expression level of FasL mRNA in the ASA group and the ASS group was higher than that in the HC group {[60.70 (8.16, 106.16)]×10 -4vs [30.14 (5.37, 78.40)]×10 -4vs [6.88 (4.57, 23.79)]×10 -4, H=18.99, P<0.001}. The expression level of LIGHTR mRNA in ASS group was lower than that in HC group {[49.79(10.75, 168.48)]×10 -4vs [15.92(3.27, 105.91)]×10 -4vs [63.47(40.85, 138.07)]×10 -4, H=11.80, P<0.001]. There was no significant difference in LIGHT mRNA among all groups ( H=4.15, P>0.05). ③Spearman correlation analysis showed that DcR3 level was positively correlated with BASDAI score and hsCRP in AS patients ( r=0.52, P<0.001; r=0.35, P<0.01), and DR3 level was negatively correlated with BASDAI score, ESR and hsCRP level ( r=-0.28, P<0.001; r=-0.25, P<0.001; r=-0.31, P<0.001). TL1A was positively correlated with BASDAI score, ESR and hsCRP level ( r=0.23, P=0.046; r=0.26, P=0.015; r=0.25, P=0.017). Conclusion:DcR3 and its signal pathway-related molecules are differentially expressed in PBMCs of patients with AS, suggesting that they may participate in the occurrence and development of AS.

Preparation and characterization of Ad-ERα-36-Fc-GFP / 生物工程学报

ERα-36 is a novel subtype of estrogen receptor α which promotes tumor cell proliferation, invasion and drug resistance, and it serves as a therapeutic target. However, only small-molecule compounds targeting ERα-36 are under development as anticancer drugs at present. Gene therapy approach targeting ERα-36 can be explored using recombinant adenovirus armed with decoy receptor. The recombinant shuttle plasmid pDC316-Ig κ-ERα-36-Fc-GFP was constructed via genetic engineering to express an Ig κ-signaling peptide-leading secretory recombinant fusion protein ERα-36-Fc. The recombinant adenovirus Ad-ERα-36-Fc-GFP was subsequently packaged, characterized and amplified using AdMaxTM adenovirus packaging system. The expression of fusion protein and functional outcome of Ad-ERα-36-Fc-GFP transduction were further analyzed with triple-negative breast cancer MDA-MB-231 cells. Results showed that the recombinant adenovirus Ad-ERα-36-Fc-GFP was successfully generated. The virus effectively infected MDA-MB-231 cells which resulted in expression and secretion of the recombinant fusion protein ERα-36-Fc, leading to significant inhibition of EGFR/ERK signaling pathway. Preparation of the recombinant adenovirus Ad-ERα-36-Fc-GFP provides a basis for further investigation on cancer gene therapy targeting ERα-36.

Baicaleininhibitsthe biological behavior of hepatocellular carcinoma stemcells by downregulation of decoy receptor 3 expression / 中国组织工程研究

BACKGROUND: To date, there is no report on the anti-hepatoma mechanism of Chinese herbal medicine targeting hepatocellular carcinoma stem cells; therefore, relevant research is necessary. OBJECTIVE: To observe the effect of baicalein on the expression of Decoy receptor 3 in hepatocellular carcinoma stem cells as well as the biological behavior of hepatocellular carcinoma stem cells after down-regulation of Decoy receptor 3.METHODS: Hepatocellular carcinoma stem cells were obtained from hepatocellular carcinoma cell lines (purchased from the Cell Bank, Chinese Academy of Sciences, Shanghai, China), cultured and passaged. The cells were cultured in the low-glucose DMEM containing nothing (control group), 200 (high-dose group), 100 (middle-dose group) or 50 μmol/L (low-dose group) baicalein. After treatment with baicalein, RT-PCR and western blot were used to detect the expression of decoy receptor 3 at mRNA and protein levels. Cell counting kit-8, flow cytometry, and Transwell assay were used to detect the proliferation, cell cycle distribution, apoptosis and migration of hepatocellular carcinoma stem cells. RESULTS AND CONCLUSION: Compared with the control group, high-dose baicalein could significantly down-regulated the expression of decoy receptor 3 mRNA and protein in hepatocellular carcinoma stem cells. High-dose baicalein was then confirmed to be the best concentration of action. Compared with the control group, in the high-dose baicalein group, the proliferation ability of hepatocellular carcinoma stem cells decreased significantly, the percentage of S-phase and G2-phase cells increased, while the percentage of G1-phase cells decreased (P < 0.05). Compared with the control group, in the high-dose baicalein group, the apoptotic rate of hepatocellular carcinoma stem cells increased significantly, while the migration ability decreased markedly. To conclude, high-dose baicalein can inhibit a series of biological behaviors of hepatocellular carcinoma stem cells by down-regulating the expression of Decoy receptor 3, which provides an experimental basis for clinical treatment of hepatocellular carcinoma with Decoy receptor 3 as the target and hepatocellular carcinoma stem cells as the object.

Association of decoy receptors and osteoprotegerin gene polymorphisms with susceptibility and clinical phenotypes of Crohn's disease / 中华消化杂志

Objective To investigate the correlation between decoy receptor (DcR) 1,DcR2,osteoprotegerin (OPG) gene polymorphisms and susceptibility of Crohn's disease (CD) in Han population in Zhejiang province.Methods From April 2008 to July 2017,at the Department of Gastroenterology of The Second Affiliated Hospital of Wenzhou Medical University,The First Affiliated Hospital of Wenzhou Medical University,Central Hospital of Wenzhou and Renmin Hospital of Wenzhou,285 patients diagnosed as having CD were enrolled,and during the same period 572 healthy individuals who received health checkup at the Second Affiliated Hospital of Wenzhou Medical University were collected as healthy control.The single nucleotide polymorphism (SNP) of DcR1 (rs12549481),DcR2 (rs1133782) and OPG (rs3102735) were examined by SNaPshot technique.An unconditional logistic regression analysis was performed to analyze the differences in each SNP mutation alleles and genotype frequencies between CD group and control group.Furthermore,their correlation with clinicopathological features of CD and the efficacy of corticosteroid and infliximab was also evaluated.Results The frequencies of mutant allele A and genotype GA + AA of DcR2 (rs1133782) of CD group were 11.93％ (68/570) and 22.81％ (65/285),respectively,which were higher than those of healthy control group (8.22％,94/1 144;and 15.91％,91/572;odds ratio (OR) =1.513,95％ confidence interval (CI) 1.088 to 2.104,P =0.013;OR =1.562,95％ CI 1.094 to 2.230,P =0.014).However there was no statistically significant difference in the mutant allele and genotype frequencies of DcR1 (rs12549481) and OPG (rs3102735) between two groups (all P ＞ 0.05).The frequencies of mutant allele C and genotype TC + CC of DcR1 (rs12549481) in patients with stricturing CD were 13.89％ (25/180)and 27.78％ (25/90),respectively,which were lower than those of patients with non-stricturing,non-penetrating CD (27.68％,62/224 and 48.21％,54/112),and the differences were statistically significant (OR =0.421,95％ CI 0.252 to 0.705,P =0.001;OR =0.413,95％ CI 0.229 to 0.747,P =0.003).Besides,the frequencies of mutant allele A and genotypes GA + AA of DcR2 in patients with penetrating CD were 7.23％ (12/166) and 13.25％ (11/83),which were lower than those of patients with non-stricturing,non-penetrating CD (15.62％,35/224 and 30.36％,34/112),and the differences were statistically significant (OR =0.407,95％ CI 0.205 to 0.809,P =0.009;OR =0.350,95％ CI 0.165 to 0.743,P =0.005).In addition,there was no statistically significant difference in the frequencies of mutant allele and genotypes of OPG (rs3102735) among subtypes of CD with different features (all P ＞ 0.012 5).Moreover,the DcR1 (rs12549481),DcR2 (rs1133782) and OPG (rs3102735) polymorphisms were not correlated with the efficacy of corticosteroid and infliximab (all P ＞ 0.05).Conclusions DcR1 (rs12549481) mutation may be correlated with stricturing CD.DcR2 (rs1133782) mutation may be correlated with CD,especially with penetrating CD.However,the gene polymorphism of OPG (rs3102735) is not correlated with the risk of CD susceptibility.And the above gene SNP may be independent of the efficacy of corticosteroid and infliximab.

Effect of serum tumor necrosis factor receptor-6 level on prognosis of AECOPD patients with respiratory failure / 中国医师杂志

Objective To study the expression of serum tumor necrosis factor receptor-6 (TR6) [decoy receptor 3 (DcR3)] in patients with acute exacerbation of chronic obstructive pulmonary disease (AECOPD) complicated with respiratory failure,and to analyze its influence on prognosis.Methods 134 patients with AECOPD admitted to our hospital from March 2015 to March 2017 were selected as the study subjects.According to whether or not respiratory failure occurred,they were divided into respiratory failure group (65 cases) and non-respiratory failure group (69 cases).Another 60 healthy volunteers who underwent physical examination in our hospital during the same period were selected as the control group.Serum albumin (ALB),C-reactive protein (CRP),first-second forced breathing volume (FEV1％),first-second forced breathing vdume (FEV1/FVC),arterial partial oxygen pressure (PaO2) and arterial partial carbon dioxide pressure (PaCO2) were collected.Enzyme-linked immunosorbent assay (ELISA) was used to detect serum levels of interleukin-8 (IL-8),tumor necrosis factor-alpha (TNF-α) and DcR3.Results The levels of ALB,FEV1,FEV1/FVC and PaO2 in respiratory failure group were significantly lower than those in non-respiratory failure group and control group (P ＜ 0.05),and those in non-respiratory failure group were significantly lower than those in control group (P ＜ 0.05);the levels of CRP,PaCO2,IL-8,TNF-α and DcR3 in respiratory failure group were significantly higher than those in non-respiratory failure group and control group (P ＜ 0.05).The survival rate of high DcR3 level group was significantly lower than that of low DcR3 level group (P ＜ 0.05).High levels of TNF-α and DcR3 were independent risk factors for adverse prognosis of AECOPD patients with respiratory failure (P ＜ 0.05).Conclusions The high expression of DcR3 in serum of AECOPD patients with respiratory failure is an independent risk factor for adverse prognosis.

Comparision of the pharmacodynamics between different batches of recombinant decoy receptor innovative drug RC28-E in retinal vascular endothelial cells / 中华实验眼科杂志

Objective To compare the protective effects of pharmacological batch RC28-E1 and pilot batch RC28-E2 on retinal vascular endothelial cells (RF/6A) under the stimulation of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).Methods RF/6A cells were divided into normal control group,VEGF + FGF group and RC28-E1 groups with different concentrations.The optimal concentration of RC28-E1 was determined by cell counting kit-8 (CCK-8) method.Cells were divided into normal control group,VEGF+FGF group,RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group,and cultured with serum-free culture medium,serum-free culture medium containing VEGF+FGF,serum-free culture medium containing VEGF+FGF+RC28-E1,serum-free culture medium containing VEGF+FGF+RC28-E2,and serum-free culture medium containing VEGF+FGF+ conbercept,serum-free medium containing VEGF+FGF+FGF trap,respectively.Cell proliferation rate was measured by CCK-8 method,cell migration ability was detected by Transwell test,and tube formation ability was detected by Matrigel assay.Results The cell proliferation rate of 0.080 mg/ml RC28-E1 group was significantly lower than that of VEGF+FGF group (P＜0.05).The cell proliferation rate of RC28-E1 group,RC28-E2 group and FGF trap group were significantly lower than that of VEGF+FGF group (P＜0.05).The number of migrated cells in RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P=0.000).The numbers of meshes formed by retinal vascular endothelial cells in RC28-E1 group,RC28-E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P =0.003,0.001,0.009,0.018).The number of tube formation in FGF trap group was significantly higher than those in RC28-E1 group,RC28-E2 group,conbercept group and normal control group (P =0.014,0.000,0.008,0.014).Conclusions Under the stimulation of VEGF + FGF,the inhibitory effect of RC28-E on the proliferation of retinal vascular endothelial cells is greater than that of conbercept,and its inhibitory effect on the tube formation is superior to that of FGF trap.There is no significant difference in the effects of different batches of recombinant decoy receptor innovative drugs on retinal vascular endothelial cells.

Comparison of pharmacodynamics between different batches of recombinant decoy receptor innovative drug RC28-E1 and RC28-E2 in retinal angiogenesis and neovascularization and its mechanism / 中华实验眼科杂志

Objective To compare the pharmacodynamics between different batches of recombinant decoy receptor innovative drug RC28-E1 and RC28-E2 in retinal angiogenesis and neovascularization,and analyze its mechanism.Methods Sixty postnatal Day 4 (P4) C57BL/6J mice were randomly divided into normal control group,vascular endothelial growth factor (VEGF)+fibroblast growth factor 2 (FGF2) group,VEGF+FGF2+RC28-E1 group,VEGF+FGF2+RC28-E2 group,VEGF+FGF2+conbercept group and VEGF+FGF2+FGF trap group by using a random number table,with 10 mice in each group.The mouse retinal explant culture system was established,and stimulated with the corresponding factors and drugs prepared in the starving culture media.The normal controls were treated with the starving media.Then the retinal explants were stained with Isolectin B4 and imaged.The number of filopodia per vascular length was quantified.In addition,ninety-six P7 C57BL/6J mice were randomly divided into normal control group,oxygen-induced retinopathy (OIR) model control group,OIR + RC28-E1 group,OIR + RC28-E2 group,OIR+conbercept group and OIR+FGF trap group by using a random number table,with 16 mice in each group.The normal controls were raised under normoxia for 10 days,and the rest of the groups were raised under hyperoxia for 5 days,then returned to normoxia for another 5 days.On P17,the retinas were isolated and stained with Isolectin B4.The stained retinas were mountedon the slides and photographed.The relative vessel obliteration and neovascularization in retina were analyzed with computer software.Then the protein levels of VEGF and FGF2 were examined by Western blot in the retinas of each group in the OIR experiment.Finally,in the RF/6A cells stimulated with VEGF and FGF2,the activities of the signaling pathways,including MEK-extracellular regulated protein kinases (Erk),protein kinase C (PKC) and protein kinase B (Akt) pathways,were examined by Western blot.All experimental procedures were evaluated and approved by the Institutional Animal Care and Use Committee of Tianjin Medical University (SYXK 2009-0001),and were in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.Results The results of retinal explant cultures showed that the numbers of filopodia per vascular length in VEGF + FGF2 + RC28-E1,VEGF + FGF2 + RC28-E2,VEGF + FGF2 + conbcrcept,and VEGF+FGF2+FGF trap groups were all significantly less than that in the VEGF+FGF2 group (all at P ＜ 0.001).The filopodia number in retinal vascular front in RC28-E1 group was similar to that in the RC28-E2 group (P =0.15),whereas the filopodia numbers in both groups were significantly decreased as compared to those in VEGF+ FGF2+conbercept group and VEGF+FGF2+FGF trap group (all at P＜0.001).The results from the OIR mouse model showed that the relative vessel obliteration area in OIR model control group was dramatically higher than those in the drug intervention groups (all at P＜0.05).There was no statistical significance in the relative vessel obliteration area between OIR+RC28-E1 group and OIR+RC28-E2 group (P =0.17),while the obliteration areas in both RC28-E-intervened groups were significantly lower than those in the OIR+conbercept group and OIR+FGF trap group (all at P＜0.05).The relative neovascular pixels in the intervention groups were significantly lower than those in the OIR model control group (all at P＜0.001).The neovascular pixels in OIR+RC28-E1 group were significantly lower than those in VEGF+FGF2+conbercept group and VEGF + FGF2 + FGF trap group (both at P ＜ 0.05),but comparable to those in OIR+RC28-E2 group (P =0.39).Western blot result showed that,the protein expression of VEGF and FGF2 in the OIR mouse retinas were significantly upregulated compared to those in the normal ones (both at P＜0.001).The upregulation of both genes were normalized by both RC28-E1 and RC28-E2.In addition,the stimulation of VEGF and FGF2 induced an enhanced activity in MEK-Erk pathway in RF/6A cells,whereas RC28-E1 inhibited the overactivation.Conclusions RC28-E1 and RC28-E2 both can inhibit angiogenesis in the retinal explants isolated from neonatal mice;they also reduce vessel obliteration and mitigate neovascularization in the OIR mouse model.Therefore,the pharmacology batch and pilot test batch of RC28-E have similar efficacies and reliable stability,and are superior in the anti-angiogenic and anti-neovascular efficacy to the currently clinically available drugs conbercept and FGF trap.RC28-E1 may suppress pathological neovascularization through inhibiting the overactivation of MEK-Erk pathway in retinal vascular endothelial cells.

Establishment of quantificative ELISA for determination of human decoy receptor 3 / 临床检验杂志

Objective To establish an ELISA for quantitative determination of decoy receptor 3 (DcR3) in human plasma.Methods A solid phase double antibody sandwich method was established for quantitative determination of DcR3.The anti-DcR3 antibody was immobilized onto ELISA plate.DcR3 in samples was captured by anti-DcR3 on ELISA plate and then detected by biotin-anti-DcR3 and subsequent peroxidase-labeled streptavidin,and the color was developed by adding substrate.The standard DcR3 samples on the same plate were detected simultaneously to calculate the DcR3 concentrations in unknown samples.The sensitivity,specificity,precision,recovery,linearity and DcR3 range in normal human adults were assessed.Results The sensitivity of the developed assay was 0.051 ng/mL.The intra-coefficient of variation (CV) was less than 10％ and inter-CV was less than 15％.The average recovery rate was 90.50％.When 2-fold amount of anti-TNF-α was added into the coated antibodies,10-fold amount of biotin-labeled anti-LIGHT,antiFAS or anti-TNF-α was added into the detection antibodies,or 10 fold amount of purified LIGHT protein was added into the standard DcR3 samples as competitor,no disturbing effects on standard curve were found.The linear range of the assay was from 0.25 to 16 ng/mL (r≥0.98).The concentration of DcR3 tested in 128 plasma samples from healthy adults was (0.21 ± 0.05) ng/mL with 95％ CI ranged from 0.14 to 0.28 ng/mL and no difference of age and sex was found.Conclusion The established ELiSA for determining plasma DcR3 exhibited high specificity,sensitivity,precision,fine linearity and wide detecting range.This method could be used for quantification of DcR3 in plasma.

Comparision of the pharmacodynamics between different batches of recombinant decoy receptor innovative drug RC28-E in retinal vascular endothelial cells / 中华实验眼科杂志

Objective To compare the protective effects of pharmacological batch RC28.E1 and pilot batch RC28.E2 on retinal vascular endothelial cells ( RF/6A) under the stimulation of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). Methods RF/6A cells were divided into normal control group, VEGF + FGF group and RC28.E1 groups with different concentrations. The optimal concentration of RC28.E1 was determined by cell counting kit.8 (CCK.8) method. Cells were divided into normal control group,VEGF+FGF group, RC28.E1 group,RC28.E2 group,conbercept group and FGF trap group,and cultured with serum.free culture medium, serum.free culture medium containing VEGF+FGF,serum.free culture medium containing VEGF+FGF+RC28.E1, serum.free culture medium containing VEGF+FGF+RC28.E2,and serum.free culture medium containing VEGF+FGF+conbercept,serum.free medium containing VEGF+FGF+FGF trap,respectively. Cell proliferation rate was measured by CCK.8 method, cell migration ability was detected by Transwell test, and tube formation ability was detected by Matrigel assay. Results The cell proliferation rate of 0. 080 mg/ml RC28.E1 group was significantly lower than that of VEGF+FGF group (P<0. 05). The cell proliferation rate of RC28.E1 group,RC28.E2 group and FGF trap group were significantly lower than that of VEGF+FGF group (P<0. 05). The number of migrated cells in RC28.E1 group,RC28.E2 group,conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group (P=0. 000). The numbers of meshes formed by retinal vascular endothelial cells in RC28.E1 group,RC28.E2 group, conbercept group and FGF trap group were significantly lower than that in VEGF+FGF group ( P=0. 003,0. 001, 0. 009,0. 018). The number of tube formation in FGF trap group was significantly higher than those in RC28.E1 group,RC28.E2 group, conbercept group and normal control group ( P = 0. 014, 0. 000, 0. 008, 0. 014 ). Conclusions Under the stimulation of VEGF+FGF,the inhibitory effect of RC28.E on the proliferation of retinal vascular endothelial cells is greater than that of conbercept,and its inhibitory effect on the tube formation is superior to that of FGF trap. There is no significant difference in the effects of different batches of recombinant decoy receptor innovative drugs on retinal vascular endothelial cells.

The infection process of BK virus in transplant recipients after renal transplantation / 中华泌尿外科杂志

Objective To investigate the characteristics and manifestations of the different stages of BK virus infection in the recipients after renal transplantation.Methods A retrospective survey from January 2015 to December 2016 was done in our hospital.A total 135 recipients were included and accepted BK virus detection in 1,3,6,9,12,15 months respectively after renal transplantation.The prevalence of decoy cell,BK virus DNA load in urine and BK virus DNA load in blood was 56 cases (41.5％),9 cases (43.7％) and 30 cases (22.2％),5 cases of BK vims nephropathy confirmed by pathological biopsy (3.7％).At the same time,51 cases (37.8％) were combined with decoy cells and virus DNA load in urine.Positive decoy cells and negative BK virus DNA load in urine was 5 cases,and Positive BK virus DNA load in urine and negative decoy cells was 8 cases.The recipients were divided into positive group of urine decoy cell,positive group of urinary BK virus DNA load,and positive group of blood BK virus DNA load.Statistical correlation analysis was conducted on the laboratory test results of the 3 groups.Results The positive group of blood BK virus DNA load were detected the high level urine decoy cell count [median of 23/10HPF(2-48/10HPF)] and high level of urinary BK virus DNA load [4.52 × 106 copies/ml (6.51 × 103-7.89 × 109 copies/ml)],significantly higher than the positive group of decoy cells [8/10HPF(2-40/10HPF)] and the positive group of urine BK virus DNA load [4.56 × 105 copies/ml(5.62 × 103-7.89 ×109 copies/ml)] (P ＜ 0.05).The decoy cell count and urine DNA load has a significant linear correlation in viruria recipients,and the urinary BK DNA load and blood BK virus DNA load has the same significant 0.939 and 0.702 in 3 months,0.969 and 0.910 in 6 months,0.782 and 0.766 in 9 months,0.898 and 0.615 in 12 months after renal transplantation.Conclusions There is a linear correlation between decoy cell in urine,viruria and viremia,suggesting that the infection of BK virus in kidney transplant recipients is a continuous process.linear correlation in viremia recipients(P ＜ 0.05).The correlation coefficients at different time points were

Regression analysis to select native-like structures from decoys of antigen-antibody docking / 生物工程学报

Given the increasing exploitation of antibodies in different contexts such as molecular diagnostics and therapeutics, it would be beneficial to unravel properties of antigen-antibody interaction with modeling of computational protein-protein docking, especially, in the absence of a cocrystal structure. However, obtaining a native-like antigen-antibody structure remains challenging due in part to failing to reliably discriminate accurate from inaccurate structures among tens of thousands of decoys after computational docking with existing scoring function. We hypothesized that some important physicochemical and energetic features could be used to describe antigen-antibody interfaces and identify native-like antigen-antibody structure. We prepared a dataset, a subset of Protein-Protein Docking Benchmark Version 4.0, comprising 37 nonredundant 3D structures of antigen-antibody complexes, and used it to train and test multivariate logistic regression equation which took several important physicochemical and energetic features of decoys as dependent variables. Our results indicate that the ability to identify native-like structures of our method is superior to ZRANK and ZDOCK score for the subset of antigen-antibody complexes. And then, we use our method in workflow of predicting epitope of anti-Ebola glycoprotein monoclonal antibody-4G7 and identify three accurate residues in its epitope.

Inhibition of Allergic Response by Intranasal Selective NF-κB Decoy Oligodeoxynucleotides in a Murine Model of Allergic Rhinitis

PURPOSE: It remains unknown whether local inhibition of Nuclear factor-kappa B (NF-κB) could have therapeutic value in the treatment of allergic rhinitis (AR). This study aimed to evaluate the effect of selective NF-κB inhibition using NF-κB decoy oligodeoxynucleotides (ODNs) for the local treatment of AR in ovalbumin (OVA)-sensitized wild-type mice. METHODS: BALB/c mice were sensitized with OVA and alum, and then challenged intranasally with OVA. NF-κB decoy ODNs were given intranasally to the treatment group, and NF-κB scrambled ODNs were given to the sham treatment group. Allergic symptom scores, eosinophil infiltration, cytokine levels in the nasal mucosa, nasal lavage fluid, and spleen cell culture, serum total and OVA-specific immunoglobulins, as well as intercellular adhesion molecure-1 (ICAM-1) in the nasal mucosa, were analyzed. RESULTS: NF-κB decoy ODNs significantly reduced allergic symptoms and eosinophil infiltration in the nasal mucosa. They also suppressed serum levels of total IgE, OVA-specific IgE, and IgG1. IL-5 and TNF-α levels and the expression of ICAM-1 were decreased in the nasal mucosa of the treatment group compared to the positive control and sham treatment groups. In addition, IL-6 levels were significantly decreased in the nasal lavage fluid of the treatment group. Furthermore, NF-κB decoy ODNs significantly reduced expression of the systemic Th2 cytokines, IL-4 and IL-5 in spleen cell culture. CONCLUSIONS: This study demonstrates for the first time that local NF-κB inhibition using NF-κB decoy ODNs suppressed the allergic response in a murine AR model. This shows the therapeutic potential of local NF-κB inhibition in the control of AR.

STAT3 Activation Way Blocking Effects on Colorectal Cancer cells HT29 Growth / 中国现代普通外科进展

Objective:Explore decoy oligonucleotide (ODN) technology targeted blocking STAT3 activation way effects on colorectal cancer HT29 cell growth,to provide experimemt basis for gene therapy of colorectal cancer.Methods:Using decoy ODN in vitro transfect colorectal cancer HT29 cell line,to targeted blocking STAT3 activation way.Fluorescence microscope and flow cytometry detect transfection conditions and efficiency,and MTT method detect the cell growth inhibition,Realtime RT-PCR and Western blotting test STAT3 in cells,the Bcl-xl and Caspase3 mRNA and protein expression.Results:STAT3 decoy ODN can efficient transfection into colorectal cancer cells,and is due to the nucleus.HT29 cell in the STAT3 decoy ODN,growth inhibition rate increased significantly,and mRNA and quantity of protein expression of STAT3 significantly reduce (P＜0.05),while caspase3 increase significantly (P＜0.05).Conclusion:Named Decoy ODN targeted after blocking STAT3 activation way,which can effectively cut colorectal cancer cells HT29 STAT3 gene expression,and inhibit the growth of the cells.The mechanism may be related to lower the expression of STAT3 and the Bcl-xl and Caspase3.

A tissue microarray study of toll‑like receptor 4, decoy receptor 3, and external signal regulated kinase 1/2 expressions in astrocytoma.

Introduction: Decoy receptor 3 (DcR3) functions as a death decoy inhibiting apoptosis mediated by the tumor necrosis factor receptor family. It is highly expressed in many tumors and its expression can be regulated by the MAPK/ ERK signaling pathway and ERK is a vital member of this pathway. Toll‑like receptor 4 (TLR4) is expressed on immune cells. Increased TLR4 expression has been associated with various types of cancers. Material and Methods: The study was conducted to investigate the expression of DcR3, ERK1/2, and TLR4 in astrocytomas and evaluate if they are validating markers for discriminating glioblastoma from anaplastic astrocytoma in limited surgical specimen. Expression of DcR3, ERK1/2, and TLR4 was determined by immunohistochemical staining of tissue microarray from 48 paraffin‑embedded tissues. A binary logistic regression method was used to generate functions that discriminate between anaplastic astrocytomas and glioblastomas. Results: The expression of TLR4 and DcR3 was significantly higher in glioblastomas than in anaplastic astrocytomas. DcR3 could discriminate anaplastic astrocytomas from glioblastomas with high sensitivity (93.8%), specificity (90%), and accuracy (92.3%). Conclusion: Our results suggest that DcR3 may be a useful marker for discriminating anaplastic astrocytomas from glioblastomas.

Infección por Poliomavirus en pacientes trasplantados renales del Hospital Carlos Andrade Marín, enero 2013 a diciembre 2014 / Polyomavirus infection in kidney transplant patients of the Carlos Andrade Marin Hospital from January 2013 to December 2014

Introducción: La nefropatía del poliomavirus tipo BK, o nefropatía por VBK, se asocia a disfunción del injerto renal teniendo como factor de riesgo del donante, la presencia de virus VBK activo, infección por Citomegalovirus. La presencia de células de Decoy en orina y de virus BK en el plasma son los marcadores de la replicación del virus del polioma. Materiales y métodos: Estudio retrospectivo observacional en pacientes sometidos a trasplante renal en el período: enero 2013 a diciembre 2014. Los métodos empleados para establecer el diagnóstico incluyeron la determinación de las características clínicas; el hallazgo de células de Decoy en orina; reacción en cadena de polimerasa (PCR) en sangre para detectar el virus BK; inmunohistoquímica en biopsia renal. Resultados: De 53 pacientes con trasplante renal, cinco desarrollaron nefropatía inducida por virus del polioma humano. De ellos, cuatro tuvieron donante cadavérico y uno, donante vivo relacionado. El deterioro de la función renal se estableció 3 a 7 meses postrasplante, en pacientes que recibieron terapia inmunosupresora a base de micofenolato de mofetilo o tacrolimus. De los cinco pacientes en uno se evidenció células de Decoy en orina positivo y todos tenían carga viral plasmática positiva. Discusión: La nefropatía por Virus BK en pacientes trasplantados renales constituye una infección secundaria relacionada con el rechazo, el diagnóstico se establece con el análisis de marcadores específicos.

Introduction: Polyomavirus type BK (BKV) nephropathy is associated with renal transplant dysfunction, since donos carrying the VBK hold a risk factor of the concurrent cytomegalovirus infection. The presence of Decoy cells in urine and the VBK in plasma are the markers of polioma virus replication. Methods: Retrospective observational study focused on patients who received renal transplants in the period between January 2013 to December 2014. In order to make the diagnosis more accurate, we tested for clinical characteristics; the presence of Decoy cells in urine; polymerase chain reaction (PCR) blood test to detect the BK virus and immune histochemical test on renal biopsies. Results: Of 53 renal transplant patients, five developed nephropathy induced by human polyoma virus. Four received transplants from cadaveric donors and one from a living donor. Renal function deterioration was seen between three to seven months after the renal transplant in those patients receiving immunosuppression with mycophenolate mofetil or tacrolimus. Of the five patients, one tested positive for decoy cells in urine, and they all tested positive for plasmatic viral load. Discusion: The presence of BKV nephropathy in renal transplant patients is a secondary infection that might cause organ rejection. The diagnosis is made with specific biomarkers.

Enhanced Anti-tumor Reactivity of Cytotoxic T Lymphocytes Expressing PD-1 Decoy

Programmed death-1 (PD-1) is a strong negative regulator of T lymphocytes in tumor-microenvironment. By engaging PD-1 ligand (PD-L1) on tumor cells, PD-1 on T cell surface inhibits anti-tumor reactivity of tumor-infiltrating T cells. Systemic blockade of PD-1 function using blocking antibodies has shown significant therapeutic efficacy in clinical trials. However, approximately 10 to 15% of treated patients exhibited serious autoimmune responses due to the activation of self-reactive lymphocytes. To achieve selective activation of tumor-specific T cells, we generated T cells expressing a dominant-negative deletion mutant of PD-1 (PD-1 decoy) via retroviral transduction. PD-1 decoy increased IFN-γ secretion of antigen-specific T cells in response to tumor cells expressing the cognate antigen. Adoptive transfer of PD-1 decoy-expressing T cells into tumor-bearing mice potentiated T cell-mediated tumor regression. Thus, T cell-specific blockade of PD-1 could be a useful strategy for enhancing both efficacy and safety of anti-tumor T cell therapy.

Effect of decoy receptor 3 gene on hepatocyte apoptosis / 临床肝胆病杂志

ObjectiveTo investigate the effect of decoy receptor 3 (DcR3) gene on hepatocyte apoptosis, as well as the possible mechanism of action of DcR3 in this process. MethodsThe human liver cell lines were cultured in vitro, and pEF1α-DcR3 transfection group, pEF1α-IRES transfection group, and negative control group were established. The pEF1α-IRES-DsRed-Express2-DcR3 eukaryotic expression vector was constructed and transfected into human liver cell lines for 36 hours. qRT-PCR was used to measure the mRNA expression of DcR3, Fas ligand (FasL), α-smooth muscle actin (α-SMA), and transforming growth factor-β1 (TGF-β1), Western blot was used to measure the change in the protein expression of DcR3, and flow cytometry was used to measure apoptosis. An analysis of variance was used for comparison of continuous data between groups, and the least significant difference t-test was used for comparison between any two groups. ResultsAfter human liver cell lines were transfected with pEF1α-DcR3 for 36 hours, the pEF1α-DcR3 transfection group showed significant increases in the mRNA and protein expression of DcR3 compared with the pEF1α-IRES transfection group and negative control group (F=33 1695 and 14154, all P＜0.01). Compared with the other two groups, the pEF1α-DcR3 transfection group showed significant reductions in the mRNA expression of FasL, α-SMA, and TGF-β1 (F=269 4518, 20 7904, and 80678, all P＜0.01), which suggested that DcR3 inhibited the expression of FasL, α-SMA, and TGF-β1. Compared with the pEF1α-IRES transfection group and negative control group, the pEF1α-DcR3 transfection group showed a significant reduction in apoptosis rate (F=55863, all P＜0.01). ConclusionDcR3 can inhibit hepatocyte apoptosis and downregulate the mRNA expression of FasL, α-SMA, and TGF-β1.

Effect of RNA DcR3 interference on the proliferation of ovarian cancer cell CAOV3 / 实用医学杂志

Objective To investigate the effects of siRNA targeting decoy receptor 3 on the cell proliferation of ovarian carcinoma cell CAOV3. Methods We constructed siRNA targeting decoy receptor 3,which was transfected into ovarian carcinoma cells CAOV3 , and observed the effects of DcR3 siRNA on the cell proliferation of CAOV3 cell by MTT experiment. The experiment contained 3 groups, including the normal control group (CAOV3 cell was not transfected), the negative control group (CAOV3 cell was transfected with blank vector) and the experimental group (CAOV3 cell was transfected with DcR3 siRNA). The expression levels of DcR3 mRNA were detected by Real-time PCR. Results DcR3 siRNA recognized and degraded DcR3 mRNA in CAOV3 cells of the experimental group. DcR3 mRNA of the experimental group was significantly decreased. The proliferation of CAOV3 cell was significantly decreased by DcR3 siRNA comparing with the normal control group and negative control group (P < 0.01). Conclusion DcR3 siRNA can inhibit the proliferation of ovarian cancer cell line CAOV3 by recognized and degraded DcR3 mRNA.

Effect of mAb2G4-ODN-lip on hypoxia-reoxygenation injury in H9c2 cardiomyocytes / 中华麻醉学杂志

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.

Targeted blocking STAT3 enhances sensitivity of liver cancer cell H22 to chemo-therapy drug doxorubicin / 中国免疫学杂志

Objective:To investigate the theraputic effect of STAT3 Decoy-ODN combined with chemotherapy drugs for HCC commonly used in clinical,include doxorubicin (DOX),5-fluorouracil (5-Fu) and cisplatin;and,analyzing the impact of combination therapy on the immune system.Methods:MTT assay was used to detect cell proliferation,and Annexin-V /7AAD double staining assay was used to detect the apoptosis of Decoy ODN transfected-hepatoma cells treated with chemotherapy drugs.The tumor growth and survival rate of H22 tumor-bearing mice treated with DOX combined with STAT3 Decoy-ODN or not were observed.FACS was applied to analyze the subpopulation and activation of PBMCs from tumor-bearing mice treated as above,and to evaluate the influence of DOX or DOX-treated tumor cells on spleen lymphocyte activation.Results: DOX-induced the suppression and the apoptosis of H22 were significantly increased by Decoy ODN transfection.The combination treatment of Decoy ODN and DOX significantly reduced H22 tumor growth and extended the survival of tumor-bearing mice.Low-dose DOX could increase the proportion of T cells and CD69+T cells in PBMCs,as well as the expression of CD107a and IFN-γin NK cells.DOXt-reated H 22 cells increased the proportion of T cells.Conclusion:Targeted blocking STAT3 could enhance the sensitivity of liver cancer cells to doxorubicin.So,combination therapy may improve DOX therapeutic effect and reduce DOX-mediated side effects.Furthermore,low dose of DOX can promote the activation of host immune system by acting on tumor cells.