Objective To investigate the effects of histone modification on the expression of chemokines in alveolar epithelial typeⅡ cells ( AECⅡ) in a rat model of chronic obstructive pulmonary disease ( COPD) . Methods 20 SD rats were randomly assigned to a normal control group and a COPD group. The rat model of COPD was established by cigarette smoking. Lung histological changes were observed by HE staining. AECⅡ cells were isolated and identified by alkaline phosphatase staining and electron microscopic. The mRNA expressions of monocyte chemoattractant protein ( MCP) -1, IL-8, and macrophage inflammatory protein ( MIP) -2αwere detected by real-time quantitative PCR. The expression of histone deacetylase ( HDAC) 2 was measured by western blot. Chromatin immunoprecipitation ( ChIP) was used todetect H3 and H4 acetylation, and H4K9 methylation in the promoter region of chemokine gene. Results Compared with the control group, the mRNA expressions of MCP-1, IL-8, and MIP-2αin the COPD group increased 4. 48,3. 14, and 2. 83 times, respectively. The expression of HDAC2 protein in the COPD group wassignificantly lower than in the control group ( 0. 25 ±0. 15 vs. 0. 66 ±0. 15, P lt; 0. 05) . The expression of HDAC2 had a negative correlation with the gene expressions of IL-8, MCP-1, and MIP-2α( r = - 0. 960,- 0. 914, - 0. 928, respectively, all P lt;0. 05) . The levels of H3 and H4 acetylation were higher, and H4K9 methylation level was lower in the promoter region of chemokine gene in the COPD group compared with the control group ( all P lt; 0. 05) . Conclusions MCP-1, IL-8, and MIP-2α participate and promote the lung inflammatory response in COPD. HDAC2-mediated histone modification may play an important role in COPD inflammation.
ObjectiveTo investigate the relationship between the pathological and functional changes of the retina and the expression of monocyte chemoattractant protein (MCP)-1 after retinal laser injury in mice. MethodsA total of 116 C57BL/6 mice were randomly divided into the normal group (58 mice) and the injured group (58 mice). Retinal laser injuries were induced by Argon ion laser. At 1, 3, 7 days after laser injury, electroretinogram (ERG) responses were recorded to detect the function of the retina. Hematoxylin and eosin (HE) staining was performed to observe pathological changes. Quantitative real-time polymerase chain reaction (PCR) was performed to detect gene expression of MCP-1. Western blot was used to measure the protein expression of MCP-1. ResultsHE staining showed a progressive damage of the retinal structure. The results of ERG showed that the differences of dark-adaptive a wave (t=6.998, 9.594, 13.778) and b wave (t=12.089, 13.310, 21.989) amplitudes of 1, 3 and 7 day post-injury between normal group and injured group were statistically significant (P=0.000). At 1 day post-injury, the differences of light adaptive b wave amplitudes between the two groups were statistically significant (t=8.844, P=0.000). While the differences of light-adaptive a wave amplitudes were not (t=2.659,P=0.200). At 3, 7 days post-injury, the differences of a (t=3.076, 7.544) and b wave amplitudes (t=10.418, 8.485) between the two groups were statistically significant (P=0.000). In dark-adaptive ERG, the differences of a wave amplitudes between 1 day and 3 days (t=3.773), 1 day and 7 days (t=5.070) and b wave amplitudes between 1 day and 7 days (t=4.762) were statistically significant (P<0.01), while the differences of a wave amplitudes between the 3 days and 7 days (t=1.297) and b wave amplitudes between 1 day and 3 days (t=2.236), 3 day and 7 days (t=2.526) were not significant (P=0.660, 0.120, 0.060). In light-adaptive ERG, the differences of a wave amplitudes between 1 day and 7 days (t=2.992) and b wave amplitudes between 1 day and 3 days (t=3.570), 1day and 7 days (t=4.989) were statistically significant (P<0.05), while the differences of a wave amplitudes between 1 day and 3 days (t=0.516), the 3 days and 7 days (t=2.475) and b wave amplitudes between 3 days and 7 days (t=1.419) were not significant (P=1.000, 0.710, 0.070). Quantitative real-time PCR showed that the differences of MCP-1 gene expression at 1, 3 and 7 day post-injury between normal group and injured group were statistically significant (t=14.329, 16.861, 5.743; P<0.05). Western blot showed that the differences of MCP-1 protein expression at 1, 3 and 7 day post-injury between normal group and injured group were statistically significant (t=75.068, 54.145, 14.653; P<0.05). ConclusionIn the first 7 days after mice retinal laser injury, there are progressive pathological and functional damage of the retina, which might be correlated with MCP-1 expression.
Objective To observe the influence of triamcinolone acetonide (TA) on the expression of pigment epitheliumderived factor (PEDF) of human retinal pigment epithelial (RPE) cells. Methods Cultured humanRPE cells (4th－6th generations) were treated with four different concentrations of TA (40, 400, 4times;103 and 4times;104 mu;g/L) for three different periods (12 or 24 or 48 hours), the levels of PEDF protein in the cell culture supernatant and cell lysates were determined by Western blot. After the initial experiment, RPE cells were treated with or without tumor necrosis factor-alpha; (TNF-alpha;, 20 ng/ml） for 24 hours, followed by TA (400 mu;g/L) treatment. The levels of PEDF and phospho-p38 mitogen activated protein kinase(p-p38MAPK) protein expression in cell culture supernatant and cell lysates were measured by Western blot. Results TAtreated RPE cells had higher PEDF expression, and 400 mu;g/L TA group had the highest effect (F＝16.98,P＜0.05). 400 mu;g/L TA treatment for one, six or 24 hours, with or without TNF-alpha; pretreatment, could all promote the PEDF expression and inhibit the p-p38MAPK protein expression (F＝16.87, 10.28; P＜0.01). TNF-alpha; pretreatment alone could inhibit PEDF protein expression and promote p-p38MAPK protein expression (F=16.87, 10.28; P＜0.01). Conclusions TA can up-regulate the expression of PEDF, and downregulate the expression of p-p38MAPK in the cultured human RPE cells.
Objective To investigate the expression of eotaxin-1, eotaxin-2 and eotaxin-3 in ARPE-19 human RPE cells after exposure to light. Methods Cultured human RPE cells (5th~10th generations) were divided into lightinduced group and control group. Cells light-induced group were exposed to the blue light at the intensity of (600plusmn;100) Lux for 12 h to establish the light damaged model. Eotaxin-1, eotaxin-2 and eotaxin-3 mRNA and protein were determined by real time polymerase chain reaction and Western blot at 0, 3, 6, 12, 24 hours after light-induced. Results In light-induced groups, mRNA levels of eotaxin-1 and eotaxin-2 were increased at 0 h (t1=6.05.t2=12.561) and 3 h （t1=2.95.t2=3.67） significantly(P＜0.05), but the mRNA level of eotaxin-3 had not changed （t3=1.57 and 1.00 respectively，P＞0.05） at that time. At 6 h （t1=4.73，t2=18.64，t3=28.48）, 12 h （t1=3.11，t2=20.62，t3=18.50）, 24 h （t1=8.25，t2=38.27，t3=18.60）, mRNA levels of eotaxin-1, 2, 3 were increased significantly （P＜0.05）. Except for the eotaxin-3 protein had not changed at 3 h （t3=1.28，P＞0.05）, protein expression of eotaxin-1, 2, 3 were increased significantly （P＜0.05） at 0 h （t1=4.85，t2=5.45，t3=6..21）, 3 h （t1=5.64，t2=4.55）, 6 h （t1=31.60，t2=6.63，t3=7.15）, 12 h （t1=14.09，t2=18.22，t3=15.76）, 24 h （t1=6.96，t2=10.47，t3=12.85）. Conclusion Eotaxin-1, eotaxin-2 and eotaxin-3 expression were increased after Light-damage, corresponding to the time after light exposure. Eotaxin-3 was the most prominent isoform.
Objective The observe the effects of interferon-inducible protein-10 (IP-10) on proliferation, migration and capillary tube formation of human retinal vascular endothelial cells (HREC) and human umbilical vein endothelial cells (HUVEC). Methods The chemokine receptor (CXCR3) mRNA of HREC and HUVEC were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). In the presence of the different concentrations of IP-10, the difference in proliferation capacity of HREC and HUVEC were analyzed by cell counting kit-8 (CCK-8) methods. Wound scratch assay and threedimensional in vitro matrigel assay were used for measuring migration and capillary tube formation of HREC and HUVEC, respectively. Results RT-PCR revealed both HREC and HUVEC expressed CXCR3. The proliferation of HREC in the presence of IP-10 was inhibited in a dosagedependent manner (F=6.202,P＜0.05), while IP-10 showed no effect on the inhibitory rate of proliferation of HUVEC (F=1.183,P＞0.05). Wound scratch assay showed a significant reduction in the migrated distance of HREC and HUVEC under 10 ng/ml or 100 ng/ml IP-10 stimulation (F=25.373, 23.858; P＜0.05). There was no effect on the number of intact tubules formed by HREC in the presence of 10 ng/ml or 100 ng/ml IP-10. The number of intact tubules formed by HREC in the presence of 1000 ng/ml IP-10 was remarkably smaller. The difference of number of intact tubules formed by HREC among 10, 100, 1000 ng/ml IP-10 and nonintervention group was statistically significant (F=5.359,P＜0.05). Conclusion IP-10 can inhibit the proliferation, migration and capillary tube formation ability of HREC and the migration of HUVEC.
Mesenchymal stem cells (MSC) are considered to have important value in the treatment of various diseases because of their low immunogenicity, transferability, and strong tissue repair capacity. Stromal cell derived factor-1 (SDF-1) and its receptor CXC chemokine receptor 4 (CXCR4) pathway plays an important role in migration of MSC. The induction of homing of MSC to retina by regulating SDF-1/CXCR4 may exert the curative effect on diabetic retinopathy to greatest exent.
ObjectiveTo investigate the expressions and significance of chemokines factor receptors 4 (CXCR4) and chemokines factor receptors 7 (CXCR7) in gastric cancer tissues. MethodsSixty-five patients with gastric cancer who treated in our hospital from January 2011 to June 2013 were retrospectively collected as gastric cancer group, and 20 patients with gastric ulcer were retrospectively collected as control group at the same time. The expressions of CXCR4 and CXCR7 in gastric cancer tissues and normal gastric tissues were measured by immunohistochemistry, and then the relation-ship among expressions of CXCR4/CXCR7 in gastric cancer tissues and clinicopathological features of patients with gastric cancer was explored, as well as its effect on survival. ResultsPositive expression rates of CXCR4 and CXCR7 were identi-fied in 80.00% (52/65) and 84.62% (55/65) of the gastric cancer group, and 5.00% (1/20) and 10.00% (2/20) in control group respectively, and the positive expression rates of CXCR4 and CXCR7 in gastric cancer group were significantly higher than those of control group respectively (χ2=36.65, P<0.01; χ2=38.55, P<0.01). The positive expression rate of CXCR4 in gastric cancer tissues was related with degree of differentiation, T staging, and TNM staging (P<0.05), positive expression rate of CXCR4 in patients with poor differentiation, T3-4 staging, and TNM Ⅲ-Ⅳ staging were higher than corresponding patients with moderate/high degree of differentiation, T1-2 staging, and TNM Ⅰ-Ⅱ staging. The positive expression rate of CXCR7 in gastric cancer tissues was related with degree of differentiation, T staging, and N staging (P<0.05), positive expression rate of CXCR7 in patients with poor differentiation, T3-4 staging, and N1-3 staging were higher than corrsponding patients with moderate/high degree of differentiation, T1-2 staging, and N0 staging. The survival situation was worse in patients with positive expression of CXCR4 and CXCR7 than corresponding patients with negative expression (P=0.01, P=0.01) respectively. ConclusionsCXCR4 and CXCR7 are related to gastric cancer genesis and development. Furthermore, the expressions of CXCR4 and CXCR7 could be used as markers to predict prognosis of gastric cancer. The regulation of CXCR4/chemokine ligand 12 (CXCL12) axis and CXCR7/CXCL12 axis may provide a new targeted therapy for patients with gastric cancer.
Objective To detect the single nucleotide polymorphisms ( SNPs) in the upstream promoter region of chemokine like factor ( CKLF) gene and analyze their possible associations with asthma and asthma-related phenotypes. Methods Direct Sequence of the 1553bp upstream promoter region of CKLF gene was performed in 245 Chinese Han human genomic DNAs ( 119 asthmatics and 126 controls) .The frequencies of alleles, genotypes, and haplotypes were determined and the association of these SNPs with asthma were further analyzed. Results Four novel SNPs, SNP88 ( T gt; C) , SNP196 ( T gt; C) , SNP568 ( C gt;G) , and SNP1047 ( C gt; G) were found in the promoter region of CKLF. The frequency of rare allele was 0. 168 ( SNP88C) , 0. 168 ( SNP196C) , 0. 352 ( SNP568G) and 0. 167 ( SNP1047G) , respectively.Haplotypes, their frequencies and the linkage disequilibrium coefficients between SNPs were constructed.Complete linkage disequilibrium( LDs) were observed between SNP88 and SNP196, SNP88 and SNP1047,as well as SNP196 and SNP1047, respectively ( D′=1. 000, r2 = 1. 000) . SNP568 was in partial LD with the other three SNPs ( r2 = 0. 366) . No association between asthma and the SNPs was observed. Conclusions Four SNPs in the regulatory region of CKLF in Chinese Han population were firstly identified. Although no significant correlation with asthma was revealed, the SNP and haplotype information is useful for other disease association studies in the future.
ObjectiveBased on the rat in situ perfusion system, to explore the effect of up-regulating Chemokine (C-X-C motif) receptor 4 (CXCR4) expression on bone marrow neutrophils in modulating its ECC-related rapid release. MethodsTwelve SD rats were randomly divided into fucoidan perfusion group (F, n=6) and control group (C, n=6) after in situ perfusion system establishment. Rats in F group received perfusion of fucoidan solution (total volume 6 ml, 1 h) and C group received buffer only. Femurs from two groups were dissected after one-hour perfusion and bone marrow tissues were collected. The neutrophil CXCR4 expression in two groups were compared using flowcytometry. Eighteen SD rats were randomly divided into fucoidan perfusion group (F', n=6), fucoidan and AMD-3100 perfusion group (F+AMD3100, n=6) and control group (C', n=6) after in situ perfusion system establishment. Rats received desired interventions before stimulation from ECC plasma. After that, 40-min perfusions of buffer were added and total counts of neutrophil in perfusates were compared. ResultsThe percentages of CXCR4 (+) cell and CXCR4 expression fluorescence in F group were 4.71%±0.21% and 161.3±7.8 respectively while the values were 1.11%±0.11% and 58.4±6.5 respectively in C group. Values in F group were both significantly higher than those in C group (P<0.05). The total counts of neutrophil in perfusates from F' group, F+AMD3100 and C' group were 261 393.7±12 470.6, 872 635.2±10 430.6 and 818 675.2±10 708.8, respectively. Statistically differences were observed between each other (P<0.05). ConclusionBone marrow neutrophil CXCR4 expression of SD rat could be effectively up-regulated by perfusion of fucoidan within the in situ perfusion system. ECC-plasma-stimulated bone marrow neutrophil release in rat could be inhibited by fucoidan induced up-regulation of neutrophil CXCR4 expression, and this inhibition effect could be canceled by AMD-3100 intervention.
Objective To explore the effect of dendritic cells (DCs) allergized by K-ras mutant peptide on expressions of chemokines CCL19, CCL22, and cytoskeletal protein fascin-1. Methods DCs were derived from peripheral blood in the presence of granuloceyte/macrophage-colony stimulating factor, interleukin (IL) -4 in vitro. The DCs were collected on day 7 after culture, and were divided into non-K-ras mutant peptide group (addition of RPMI 1604 culture solution 50 μg/ml) and K-ras mutant peptide group (addition of K-ras mutant peptide 50 μg/ml). Phenotype was identified by flow cytometry. The morphological structure was observed by scanning and transmission electron microscopies, respectively. The expressions of IL-12, CCL19, and CCL22 were tested continuously by enzyme-linked immunosorbent assay (ELISA). The expression of cytoskeletal protein fascin-1 was determined by Western blot. Results ①The expressions of CD1a, CD80, and CD86 after loading K-ras mutant peptide were higher than that before loading K-ras mutant peptide (Plt;0.01). ②The DCs with petal-like and branch-like profections after loading were observed under scanning electron microscopy; The DCs with irregular shapes, branch-like or burr-like were showed under transmission electron microscopy. ③The expressions of IL-12, CCL19, and CCL22 in the Kras mutant peptide group were higher than those in the non-K-ras mutant peptide group at different times (6, 12, 24, and 48 h) after loading Kras mutant peptide (Plt;0.01). ④The expression of fascin-1 in the K-ras mutant peptide group was also higher than that in the non-K-ras mutant peptide group (Plt;0.01). Conclusion K-ras mutant peptide can promote DC to mature and improve the expression of chemokines and cytoskeletal protein which will strengthen DC migration.