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.
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.
Objective To investigate the influence on matrix metalloproteinases (MMP) 3, 9, and 13 levels of human articular cartilage cells after blocking stromal cell derived factor 1 (SDF-1)/ chemokine receptor 4 (CXCR4) signaling pathway withAMD3100 and to define the function mechanism of AMD3100. Methods A total of 144 cartilage blocks from 12 osteoarthritis (OA) patients undergoing total knee arthroplasty (OA cartilage group) and 144 normal cartilage blocks (Mankin score of 0 or 1) from 12 patients undergoing traumatic amputation (normal cartilage group). OA cartilage group was further divided into subgroups A1, B1, and C1, and normal cartilage group into subgroups A2, B2, and C2. The cartilage tissues were cultured in DMEM solution containing 100 ng/mL SDF-1 and 1 000 nmol/L AMD3100 in subgroup A, 100 ng/mL SDF-1 and 1 000 nmol/L MAB310 in subgroup B, and 100 ng/mL SDF-1 in subgroup C, respectively. The levels of MMP-3, 9, and 13 were measured by ELISA; the expressions of MMP-3, 9, and 13mRNA were tested by RT-PCR. Results ELISA and RT-PCR results showed that the levels of MMP-3, 9, and 13 and the expressions of MMP-3, 9, and 13 mRNA were significantly lower in subgroup A than in subgroups B and C at the same time points (P lt; 0.05); the levels of MMP-3, 9, and 13 and the expressions of MMP-3, 9, and 13 mRNA were significantly higher in OA cartilage group than in normal cartilage group at the same time points (P lt; 0.05). Conclusion SDF-1 could induce overexpression and release of MMP-3, 9, and 13 in the articular cartilage through the SDF-1/CXCR4 signaling pathway; AMD3100 could reduce the mRNA expressions and secretion of MMP-3, 9, and 13 in OA cartilage by blocking the SDF-1/CXCR4 signaling pathway; but AMD3100 could not make the secretion of MMP-3, 9, and 13 return to normal levels in OA cartilage.
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.
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 To observe the levels of vascular endothelial growth factor (VEGF), interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in aqueous humor of patients with macular edema secondary to central retinal vein occlusion (CRVO). Methods Forty eyes of 40 consecutive patients with macular edema secondary to CRVO (CRVO group) were enrolled in this study. The patients included 25 males and 15 females. The patient age ranged from 38 to 76 years. The control group was 20 patients with senile cataract who underwent phacoemulsification, including 10 males and 10 females. The levels of VEGF165, VEGF165b, IL-6 and MCP-1 in aqueous humor were determined by enzymelinked immunosorbent assay. The correlation of VEGF, and IL-6, and MCP-1 were analyzed. Results The median aqueous level of VEGF165, IL-6 and MCP-1 were 1089.0, 165.6, 1253.0 pg/ml respectively in CRVO group, which were higher than the control group's results (168.2, 4.7, 216.4 pg/ml respectively), the differences were statistically significant (Z=-4.549, -6.008, -5.343;P＜0.001). The VEGF165b in CRVO group and control group were 834.0, 915.9 pg/ml respectively, the difference was not statistically significant (Z=-0.207，P＞0.05). The ratio of VEGF165b to VEGF165 in CRVO group and control group were 2.71, 7.28 respectively, the difference was statistically significant (t=-3.007，P＜0.05). There was a highly positive correlation between IL-6 and VEGF in CRVO group (r=0.526,P=0.001) and also mild positive correlation in control group (r=0.425，P=0.070). No correlation between MCP-1 and VEGF was observed in both groups (CRVO group: r=0.211,P＞0.05. Control group: r=-0.019,P＞0.05). Conclusions VEGF165, IL-6 and MCP-1 levels were increased in CRVO patients while the VEGF165b was normal. The ratio between VEGF165b and VEGF165 in aqueous humor of patients with macular edema secondary to CRVO was decreased.
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 observe the effects of stromal cellderived factor 1alpha; (SDF-1alpha;) in secondary neovascular glaucoma (NVG) of proliferative diabetic retinopathy (PDR). Methods The vitreous specimens from 25 PDR patients (31 eyes) were collected with 13 NVG eyes and non-NVG 18 eyes. The concentrations of SDF-1alpha; and vascular endothelial growth factor (VEGF) in those specimens were measured by enzyme-linked immunosorbent assay （ELISA）. Human umbilical vein endothelial cells (HUVEC) were treated by different concentrations of SDF-1alpha;and vascular endothelial growth factor (VEGF) in vitro, and the formation of tube cavity-like structure, length of capillarylike structures and 5prime;-bromo-2prime;-deoxyuridine (BrdU) labeling of treated HUVEC were measured. Results The length of HUVEC tube-like and capillarylike structure formation in 10, 100, 1000 ng/ml SDF-1alpha; and 10 ng/ml VEGF groups were longer than that in the control group, the differences were statistically significant (P＜0.01). The A value of BrdU labeling of 10, 100, 1000 ng/ml SDF-1alpha; and 10 ng/ml VEGF groups were increased than that in the control group, the differences were statistically significant (P＜0.01). The vitreous levels of SDF-1alpha; and VEGF of NVG specimens were higher than those in the non-NVG group, the differences were statistically significant (P＜0.01). Conclusions SDF-1alpha; may promote the migration and proliferation of vascular endothelium cells, and participate in the neovascularization process in NVG patients with PDR.
Objective To evaluate the effects of inflammatory cytokines, including tumor necrosis factorTNF-alpha; and interleukins (IL-6 and IL-8), to the expression of pigment epithelium-derived factor (PEDF) in human retinal pigment epithelium (RPE)cells. Method Cultured primary human RPE cells were treated with 20，2，0.2 , and 0.02 ng/ml of TNF-alpha;, IL-6 and IL-8 separately. The levels of PEDF expression were determined by Western blot of the supernant after 6，12，24 and 48 hours of culture. Results PEDF secretion of RPE cells was inhibited by TNF-alpha;, IL-6 and IL-8 in a time- and dose-dependent fashion. Compared with the controls, the expression of PEDF decreased significantly in 0.02 ng/ml and 6 hours group (F＝7.14, P＜0.05）, 2.00 ng/ml and 48 hours group（F=14.05，P＜0.01) , and 20.00 ng/ml and 24 hours group（F=11.53，P＜0.01). TNF-alpha; was the most strength inhibitor (F＝14,P＜0.01）.Conclusion TNF-alpha;, IL-6, and IL-8 could suppress the expression of PEDF in the cultured human RPE cells.
Objective To explore the expression of chemokine receptor CCR7 in thyroid papillary microcarcinoma tissues and the relationship with clinicopathological features. Methods The CCR7 expressions in 31 cases of thyroid papillary microcarcinoma, 34 cases of thyroid papillary carcinoma which diameter＞1cm, 34 cases of nodular goiter, and 12 cases of thyroid papillary microcarcinoma contralateral normal thyroid tissues were detected by using immunohistochemistry S-P method. Results The expression positive rates of CCR7 in thyroid papillary microcarcinoma and papillary thyroid carcinoma which diameter＞ 1cm were both 100%, the difference had not statistically significant （P＞0.05）. In nodular goiter and normal thyroid tissues, the expression positive rate of CCR7 was 64.7% and 33.3%, respectively, and compared with thyroid papillary microcarcinoma, the difference had statistically significant （P＜0.05）. There were not relations between the expression of CCR7 and patient’s gender, age, capsule invasion, and lymph node metastasis （P＞0.05）. Conclusions The CCR7 in thyroid papillary microcarcinoma and thyroid papillary carcinoma which diameter＞ 1cm are both high expressions, and have the same bionomics, both prone to cervical lymph node meta-stasis, and the radical neck dissection （central area） are both need to take.