Effects of ethanol on the properties of platelets and endothelial cells in model experiments

AIM: To investigate effects of ethanol on activity markers of atherosclerosis in an in vitro endothelial cell model. METHODS: After 24 h incubation with ethanol (0.0095%), human umbilical vein endothelial cells were stimulated for 1 h with lipopolysaccharide, and were then incubated in direct contact with activated platelets. Following this incubation, the expression of CD40L and CD62P on platelets, and the expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), urokinase plasminogen activator receptor (uPAR), and membrane-type 1 matrix metalloproteinase (MT1-MMP) on endothelial cells were measured by flow cytometry. RESULTS: The increased expression of VCAM-1 and uPAR on endothelial cells by proinflammatory stimulation with activated platelets was significantly reduced through pre-incubation with ethanol (P<0.05). Furthermore, platelets in direct contact with ethanol and with endothelial cells pre-incubated in ethanol showed a significant reduction in their CD40L expression (P<0.05). Ethanol had no significant effect on ICAM-1 and MT1-MMP expression on endothelial cells. CONCLUSION: Ethanol directly attenuates platelet activation and has significant endothelial cell-mediated effects on selected markers of atherosclerosis in vitro . These findings underline possible protective effects of ethanol on atherosclerosis.     

Cooperative inhibitory effects of antisense oligonucleotide of cell adhesion molecules and cimetidine on cancer cell adhesion

AIM:To explore the cooperative effects of antisenseoligonucleotide (ASON) of cell adhesion molecules andcimetidine on the expression of E-selectin and ICAM-1 inendothelial cells and their adhesion to tumor cells.METHODS:After treatment of endothelial cells with ASONand/or cimetidine and induction with TNF-α,the proteinand mRNA changes of E-selectin and ICAM-1 in endothelialcells were examined by flow cytometry and RT-PCR,respectively.The adhesion rates of endothelial cells to tumorcells were measured by cell adhesion experiment.RESULTS:In comparison with TNF-α inducing group,lipo-ASON and lipo-ASON/cimetidine could significantly decreasethe protein and mRNA levels of E-selectin and ICAM-1 inendothelial cells,and lipo-ASON/cimetidine had mostsignificant inhibitory effect on E-selectin expression (from36.37±1.56% to 14.23±1.07%, P<0.001).Meanwhile,cimetidine alone could inhibit the expression of E-selectin(36.37±1.56% vs 27.2±1.31%,P<0.001),but not ICAM-1(69.34±2.50% vs 68.07±2.10%,P<0.05)and the two kinds ofmRNA,either.Compared with TNF-α inducing group,the rateof adhesion was markedly decreased in lipo-E-selectin ASONand lipo-E-selectin ASON/cimetidine treated groups(P<0.05),and lipo-E-selectin ASON/cimetidine worked better thanlipo-E-selectin ASON alone except for HepG2/ECV304 group(P<0.05).However,the decrease of adhesion was notsignificant in lipo-ICAM-1 ASON and lipo-ICAM-1 ASON/cimetidinetreated groups except for HepG2/ECV304 group (P>0.05).CONCLUSION:These data demonstrate that ASON incombination with cimetidine in vitro can significantly reducethe adhesion between endothelial cells and hepatic orcolorectal cancer cells,which is stronger than ASON orcimetidine alone.This study provides some useful proofsfor gene therapy of antiadhesion.     

The Effect of Dehydroepiandrosterone on the Expression of AT1 receptor and TNF-induced ICAM-1 in Vascular Smooth Muscle Cells

Objectives To further investigate the molecular mechanism of vasoprotective role of dehydroepiandrosterone (DHEA), we examined DHEA on AT1 receptor and ICAM-1 gene expression in vascular smooth muscle cells (VSMCs). Methods RT-PCR and Western Blot was used to determine the change of the expressions of mRNA and protein of AT1 and ICAM-1 when given various concentration dehydroepiandrosterone. Results 1.AT1 was abundant under the basal condition. The expression of AT1 mRNA and protein decreased after stimulated by DHEA (at 10^-10mol/L, 10^-8mol/L, 10^-6mol/L), and the effects of DHEA on AT1 protein was dose-dependent. ER inhibitor Tamoxifen and AR inhibitor Flutamide enhanced AT1 protein expression, but did not influence the mRNA expression. 2. The exp-ression of ICAM-1 gene was low under the basal condition.It increased when induced by TNF-α, but decreased when induced by DHEA (at 10^-10mol/L, 10^-8moL/L, 10^-6mol/L) ,and the effects of DHEA on ICAM-1 gene expression were dose-dependent. Conclusions These findings suggest that DHEA modulates AT1 and inflammatory factor induced ICAM-1 gene expression in VSMC, butfurther studies are necessary in the mecha-nism of DHEA action.     

Hypoxia inducible factor-1α mediates protective effects of ischemic preconditioning on ECV-304 endothelial cells

AIM: To investigate whether hypoxia inducible factor-1α (HIF-1α) is linked to the protective effects of ischemic preconditioning (IP) on sinusoidal endothelial cells against ischemia/reperfusion injury. METHODS: Sinusoidal endothelial cell lines ECV-304 were cultured and divided into four groups: control group, cells were cultured in complete DMEM medium; cold anoxia/warm reoxygenation (A/R) group, cells were preserved in a 4℃ UW solution in a mixture of 95% N2 and 5% CO2 for 24 h; anoxia-preconditioning (APC) group, cells were treated with 4 cycles of short anoxia and reoxygenation before prolonged anoxia- preconditioning treatment; and anoxia-preconditioning and hypoxia inducible factor-1α (HIF-1α) inhibitor (I-HIF-1) group, cells were pretreated with 5 μm of HIF-1α inhibitor NS398 in DMEM medium before subjected to the same treatment as group APC. After the anoxia treatment, each group was reoxygenated in a mixture of 95% air and 5% CO2 incubator for 6 h. Cytoprotections were evaluated by cell viabilities from Trypan blue, lactate dehydrogenase (LDH) release rates, and intracellular cell adhesion molecule-1 (ICAM-1) expressions. Expressions of HIF-1α mRNA and HIF-1α protein from each group were determined by the RT-PCR method and Western blotting, respectively. RESULTS: Ischemia preconditioning increased cell viability, and reduced LDH release and ICAM-1 expressions. Ischemia preconditioning also upregulated the HIF-1α mRNA level and HIF-1α protein expression. However, all of these changes were reversed by HIF-1α inhibitor NS398.CONCLUSION: Ischemia preconditioning effectively inhibited cold hypoxia/warm reoxygenation injury to endothelial cells, and the authors showed for the first time HIF-1α is causally linked to the protective effects of ischemic preconditioning on endothelial cells.     

Expression of E-selectin, integrinβ_1 and immunoglobulin superfamily member in human gastric carcinoma cells and its clinicopathologic significance

AIM: To study the expression levels of E- selectin, integrinβ1 and immunoglobulin supperfamily member-intercellular adhesion molecule-1 (ICAM-1) in human gastric carcinoma cells, and to explore the relationship between these three kinds of cell adhesion molecules and gastric carcinoma. METHODS: The serum contents of E-selectin, integrinβ1 and ICAM-1 were detected by enzyme-linked immuno-sorbent assay (ELISA), in 47 healthy individuals (control group) and in 57 patients with gastric carcinoma (gastric carcinoma group) respectively prior to operation and 7 d after operation. RESULTS: The serum E-selectin, ECAM-1 and integrinβ1 were found to be expressed in both control and gastric carcinoma groups. However, they were highly expressed in patients with gastric carcinoma patients before operation or with unresectable tumours. The expression levels of ICAM-1 and integrinβ1 were significantly higher in gastric carcinoma patients than in controls (P < 0.01). A comparison of the E-selectin levels between the two groups showed statistically insignificant differnce (P = 0.64). In addition, the expression levels were all decreased substantially in the postoperative patients subjected to radical resection of the tumours, indicating that the high level expressions of these compounds might be the important factor for predicting the prognosis of these patients. CONCLUSION: Serum E-selectin, ICAM-1 and integrin pi expression levels are probably related to the metastasis and relapse of gastric cancer.     

Expression of adhesion molecules on mature cholangiocytes in canal of Hering and bile ductules in wedge biopsy samples of primary biliary cirrhosis

AIM: To examine the expression of intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) expression on canals of Hering (CoH) and bile ductules associated with the autoimmune process of bile duct destruction in primary biliary cirrhosis (PBC). METHODS: Ten wedged liver biopsies of PBC (five cases each of stages 2 and 3) were studied. The liver specimens were processed for transmission electron microscopy. Immunohistochemistry was performed using anti-ICAM-1 and anti-LFA-1 mouse mAbs. In situ hybridization was done to examine the messenger RNA expression of ICAM-1 in formalin-fixed, paraffin-embedded sections using peptide nucleic acid probes and the catalyzed signal amplification (CSA) technique. Immunogold-silver staining for electron microscopy was performed using anti-ICAM and anti-LFA-1 mouse mAbs. The immunogold particles on epithelial cells of bile ductules and cholangiocytes of CoH cells were counted and analyzed semi-quantitatively. Western blotting was performed to confirm ICAM-1 protein expression. RESULTS: In liver tissues of PBC patients, immunohi-stochemistry showed aberrant ICAM-1 expression on the plasma membrane of epithelial cells lining bile ductules, and also on mature cholangiocytes but not on hepatocytes in CoH. LFA-1-positive lymphocytes were closely associated with epithelial cells in bile dcuctules. ICAM-1 expression at protein level was confirmed by Western blot. In situ hybridization demonstrated ICAM-1 mRNA expression in bile ductules and LFA-1 mRNA in lymphocytes infiltrating the bile ductules. By immunoelectron microscopy, ICAM-1 was demonstrated on the basal surface of epithelial cells in bile ductules and on the luminal surfaces of cholangiocytes in damaged CoH. Cells with intermediate morphology resembling progenitor cells in CoH were not labeled with ICAM-1 and LFA-1. CONCLUSION: De novo expression of ICAM-1 both on mature cholangiocytes in CoH and epithelial cells in bile ductules in PBC implies that lymphocyte-induced destruction through adhesion by ICAM-1 and binding of LFA-1-expressing activated lymphocytes takes place not only in bile ductules but also in the CoH.     

Induction of macrophage inflammatory cytokines by Ox-LDL is ABCA1 dependent

Objective The current study aimed to evaluate whether the induction of macrophage inflammatory cytokines by Ox-LDL is related to the expression of ABCA 1 pathway. Methods After THP 1/PMA macrophages were transfected with ABCA1 antisense oligonucleotides （100nmol/L） followed by treatment with Ox-LDL （30mg/L）, the expressions of ABCA1, ICAM-1 and MCP-1 mRNA and protein were determined by real-time fluorescent quantitative RT-PCR, Western blot or ELISA. Results Ox-LDL induced expressions of ABCA1, ICAM-1, and MCP-1 at both mRNA and protein levels from THPI/PMA macrophages. Transfection with ABCAI antisense oligonucleotides reduced ABCA1 mRNA levels after 3 and 6 hours and protein levels after 12 and 24 hours. The expression of ICAM-1 and MCP-1 induced by Ox-LDL was also decreased after inhibition of ABCA 1 protein expression by ABCA 1 antisense oligonucleotide decreased. Conclusion The induction of macrophage inflammatory cytokines by Ox-LDL is partially dependent on expression ofABCA1. Our studies disclose new functions of ABCA1 in macrophages Objective The current study aimed to evaluate whether the induction of macrophage inflammatory eytokines by Ox-LDL is related to the expression of ABCA 1 pathway. Methods After THP 1/PMA macrophages were transfected with ABCA1 antisense oligonucleotides （100nmol/L） followed by treatment with Ox-LDL （30mg/L）, the expressions of ABCA1, ICAM-1 and MCP-1 mRNA and protein were determined by real-time fluorescent quantitative RT-PCR, Western blot or ELISA. Results Ox-LDL induced expressions of ABCA1, ICAM-1, and MCP-1 at both mRNA and protein levels from THPI/PMA macrophages. Transfection with ABCAI antisense oligonucleotides reduced ABCA1 mRNA levels after 3 and 6 hours and protein levels after 12 and 24 hours. The expression of ICAM-1 and MCP-1 induced by Ox-LDL was also decreased after inhibition of ABCA 1 protein expression by ABCA 1 antisense oligonucleotide decreased. Conclusion The induction of macrophage inflammatory cytokines by Ox-LDL is partially dependent on expression ofABCA1. Our studies disclose new functions of ABCA1 in macrophages     

Cooperative inhibitory effects of antisense oligonucleotide of cell adhesion molecules and cimetidine on cancer cell adhesion

AIM: To explore the cooperative effects of antisense oligonucleotide (ASON) of cell adhesion molecules and cimetidine on the expression of E-selectin and ICAM-1 in endothelial cells and their adhesion to tumor cells. METHODS: After treatment of endothelial cells with ASON and/or cimetidine and induction with TNF-α, the protein and mRNA changes of E-selectin and ICAM-1 in endothelial cells were examined by flow cytometry and RT-PCR,respectively. The adhesion rates of endothelial cells to tumor cells were measured by cell adhesion experiment. RESULTS: In comparison with TNF-α inducing group, lipoASON and lipo-ASON/cimetidine could significantly decrease the protein and mRNA levels of E-selectin and ICAM-1 in endothelial cells, and lipo-ASON/cimetidine had most significant inhibitory effect on E-selectin expression (from 36.37&#177;1.56% to 14.23&#177;1.07%, P&lt;0.001). Meanwhile,cimetidine alone could inhibit the expression of E-selectin(36.37&#177;1.56% vs 27.2&#177;1.31%, P&lt;0.001), but not ICAM-1(69.34&#177;2.50% vs68.07&#177;2.10%,P&gt;0.05)and the two kinds ofmRNA, either. Compared with TNF-α inducing group, the rate of adhesion was markedly decreased in lipo-E-selectin ASON and lipo-E-selectin ASON/cimetidine treated groups(P&lt;0.05),and lipo-E-selectin ASON/cimetidine worked better than lipo-E-selectin ASON alone except for HepG2/ECV304 group(P&lt;0.05). However, the decrease of adhesion was not significant in lipo-ICAM-1 ASON and lipo-ICAM-1 ASON/dmetidine treated groups except for HepG2/ECV304 group (P &gt;0.05). CONCLUSION: These data demonstrate that ASON in combination with cimetidine in vitro can significantly reduce the adhesion between endc~thelial cells and hepatic or colorectal cancer cells, which is stronger than ASON or cimetidine alone. This study provides some useful proofs for gene therapy of antiadhesion.     

The role of endotoxin,TNF—α,and IL—6 in inducing the state of growth hormone insensitivity

AIM：Critical illnesses such as sepsis,trauma,and burns cause a growth hormone insensitivity,wehich leads to an increased negative nitrogen balance.Endotoxin is generously released into blood under these conditions and stimulates the production of proinflammatory cytokines such as TNF-α,IL-6,and IL-1 which may play a very important role in inducing the growth hormone insensitivity,The objective of this current study was to investigate the role of endotoxin,TNFα and IL-6 in inducing the growth hormone insensityvity at the receptor and post-receptor levels.METHODS:Spageu-Dawley rats were injected with endotoxin,TNF-α,and IL-6,respectively and part of rats injected with endotoxin was treated with exogenous somatotropin simultaneously,All rats were killed at different time points,The expression of IGF-I,GHR,SOCS-3 and β-actin mRNA in the liver was detected by RT-PCR and the GH levels were measured by radioimmunoassay,the levels of TNF-α and IL-6 were detected by ELISA.RESULTS:There was no significant difference in serous GH levels between experimental group and control rats after endotoxin injection,However,liver IGF-1 mRNA expression had been obviously down-regulated in endotoxeminc rats.Liver GHR mRNA expression also had a predominant downregulation after endotoxin injection,The lowest regulation of liver IGF-I mRNA expression occurred at 12h after LPS injection,being decreased by 53% compared with control rats.For GHR mRNA expression,the lowest expression occurred at 8h and had a 81% decrease.Although SOCS-3 mRNA was weakly expressed in control rats,it was strongly up-regulated after LPS injection and had a 7.84 times increase compared with control rats.Exogenous GH could enhance IGF-1 mRNA expression in control rats,but if did fail to prevent the decline in IGF-1 mRNA expression in endotoxemic rats,Endotoxin stimulated the production of TNF-α and IL-6,and the elevated IL-6 levels was shown a positive correlation with increased SOCS-3 mRNA expression.The liver GHR mRNA expression was obviously down-regulated after TNF-α iv injection and had a 40 decresase at 8 h,but the liver socs-3 mRNA expression was the 4.94 times up-regulation occurred at 40 min after IL-6 injection.CONCLUSION:The growth homone insenstivity could be induced by LPS injection,which was associated with down regulated GHR mRNA expression at receptor level and with up-regulated SOCS-3 mRNA expression at post-receptor level The in vivo biological activities of LPS were mediated by TNF-α and IL-6 indirectly,and TNF-αand IL-6 may exert their effects on.the receptor and post-receptor levels respectively.     

Expression of toll-like receptor 4 and MD-2 gene and protein in Kupffer cells after ischemia-reperfusion in rat liver graft

AIM:To investigate the expression of toll-like receptor 4(TLR4)and MD-2 gene and protein in Kupffer cells(KCs)and their role in ischernia-reperfusion(IR)injury of ratliver graft.METHODS:KCs were isolated at 0(control group),2,12,24h(IR group)following IR in rat liver graft,mRNA expressionof TLR4 and MD-2 was detected by RT-PCR analysis,proteinexpression of TLR4/ND-2 was detected by flow cytometric(FCM)analysis,and tumor necrosis factor-α(TNF-α)levelin supernatant was measured by ELISA.Then isolated KCswere incubated with anti-TLR4 polyclonal antibody(anti-TLR4 group),and TNF-α level was measured again.RESULTS:The mRNA and protein expression of TLR4/MD-2 and the level of TNF-α in IR group increased significantlyat 2 h following IR,and reached the maximum at 12 h,andslightly decreased at 24 h,but were still significantly higherthan those in the control group(P<0.01).The expression ofthese factors was markedly decreased after anti-TLR4antibody treatment as compared with the IR group(P<0.01).CONCLUSION:Lipopolysaccharide(LPS)following IR canup-regulate TLR4/MD-2 gene and protein expression inKCs,and synthesize cytokine TNF-α.Anti TLR4 antibodycan inhibit the production of TNF-α induced by LPS.TLR4and its partner molecule MD-2 may play an important rolein Kupffer cell activation and IR injury.     

Effect of cold-ischemia time on nuclear factor-κB activation and inflammatory response in graft after orthotopic liver transplantation in rats

AIM: To study the mechanism and effect of nuclear factor-κB (NF-κB) activation and inflammatory response on the extended cold-preserved graft injury after orthotopic liver transplantation (OLT). METHODS: OLT was performed in rats with varying time of cold ischemia grafts (6, 18 and 24 h in University Wisconsin solution at 4 ℃). We determined the time of NF-κB activation and expression of tumor necrosis factor-α (TNF-α), cytokineinducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1 (ICAM-1) within 6 h after reperfusion. Serum alarming aminotransferase (ALT), neutrophil sequestration, circulating neutrophil CD11b and L- selectin expression were also evaluated. RESULTS: The accumulation of neutrophils in the graft was significantly increased in the 18 h and 24 h cold-ischemia groups within 0.5 h after reperfusion, compared with the 6 h group. But the strongly activated neutrophils was slightly increased at 2 h after reperfusion and remained at high levels 4 h after reperfusion, which was synchronized with the common situation of recipients after transplantation. Prolonged cold-preservation did not affect neutrophil accumulation and activation. NF-κB activation preceded the expression of TNF-α, CINC, and ICAM-1 in the liver, which was significantly increased with prolonged cold preservation. In prolonged cold preserved grafts, prominently elevated NF-κB activation occurred at 0.5 h and 1 h, compared with that at 2 h after reperfusion, which was consistent with greatly increased intrahepatic TNF-α response. CONCLUSION: NF-κB activation is correlated with the expression of TNF-α, CINC, and ICAM-1 in vivo in OLT rats. Extended cold preservation of grafts might up-regulate TNF-α, CINC, and ICAM-1 expression in the grafts, most probably through elevated NF-κB activation, and might contribute to neutrophil infiltration in the grafts after reperfusion. Elevated NF-κB activity is harmful to inflammatory response in the grafts, and inhibited NF-κB activity might protect against early graft injury after liver transplantation.     

Serum concentration changes of sICAM-1, hs-CRP and TNF-α after vertebral artery stenting and its clinical significance

Objective To observe the dynamic changes of serum inflammatory factors after vertebral artery stenting and to investigate its clinical significance. Methods A total of 48 patients treated with vertebral artery stenting were included, and 48 patients only received cerebral angiography were used as a control group. The levels of soluble intercellular adhesion molecule-1 (sICAM-1), high-sensitivity C-reactive protein (hs-CRP) and tumor-necrosis factor-α (TNF-α) were detected before procedure (angiography), at 24 h, 48 h, 3 d, and 1 and 3 weeks after procedure (angiography). Results The serum levels of hs-CRP (4. 85 ± 0. 53 mg/L vs. 2. 57 ±0. 36 mg/L,P＜0. 05), TNF-α (2.42 ±0. 34 μg/L vs. 1. 08 ±0. 37 μg/L,P ＜0. 05) and sICAM-1 (449.43 ± 47. 16 μg/L vs. 269. 15 ± 37. 46 μg/L, P ＜ 0. 05) at 24 hours after procedure in the stenting group were significantly elevated compared with those before procedure. The Hs-CRP level (6.24 ± 0.59 mg/L) reached the peak at 48 hours after procedure. At week 3 (2. 51 ±0.29 mg/L), it returned to the level before procedure (2. 57 ±0. 36 mg/L); TNF-α level reached the peak at day 3 (2.30 ± 0.25 μg/L), and it remained higher level at week 3 (1. 89 ±0. 13 μg/L); the sICAM-1 level continued to rise at week 3 (296. 95 ± 59. 72 μg/L). The serum hs-CRP, TNF-α and sICAM-1 levels at 24 hours after procedure in the stenting group were significantly higher than those (3. 25 ±0.40 mg/L、J. 18 ±0. 19 μg/L and 336. 57 ± 50. 18μg/L) in the control group (all P＜0.05). Conclusions The serum hs-CRP, TNF-α, sICAM-1 levels were significantly elevated after vertebral artery stenting. It was suggested that the stenting caused a longer duration of inflammatory response.     

胰岛素样生长因子对心力衰竭大鼠心肌细胞黏附分子1表达的影响

Objective To investigate the effect of insulin-like growth factor-1 (IGF-1) on intercellular adhesion molecule-1 (ICAM-1) expression in rat model with congestive heart failure (CHF). Methods The model of CHF in male Sprague-Dawley rat was established using subcutaneous injection of isoproterenol. The rats were randomized into model group (n = 10),angiotensin converting enzyme inhibitors (ACEI) group ( n=10) and IGF- 1 group ( n = 10). The rats injected with saline were used as normal controls (n=10). The haemodynamic parameters of rats in each group were detected. The concentration of plasma angiotensin Ⅱ (Ang Ⅱ ) and expression of ICAM-1 in rat myocardium were determined by radioimmunoassay and immunohistochemistry,respectively. Results Compared with model group, ACEI and IGF-1 could rescue the diversities of hemodynamic parameters. In addition, ACEI and IGF-1 could also significantly down-regulated concentration of plasma Ang Ⅱ and inhibited ICAM-1 expression. Compared with ACEI, IGF-1 more significantly inhibited ICAM-I expression (0. 804 ± 0. 024 vs. 1. 254 ± 0. 059) and down-regulated concentration of plasma AngⅡ [(369.2±65.0) μg/L vs. (384.4±56.2) μg/L]. Conclusions IGF-1 can suppress ICAM-1 expression in rat model with CHF induced by isoproterenol. This effect may be related to inhibiting activation of RAS system.     

In vivo suppressive effect of nudear factor-κB inhibitor on neutrophilic inflammation of grafts after orthotopic liver transplantation in rats

AIM. To investigate the effect of pyrrolidine dithiocarbamate (PDTC), a novel nuclear factor-κB (NF-κB) inhibitor, on expression of multiple inflammatory mediators and neutrophilic inflammation of cold preserved grafts after rat liver transplantation and its significance.METHODS: Orthotopic liver transplantation (OLT) was performed after 24 h of cold storage using University of Wisconsin solution with varied concentrations of PDTC. We determined the time course of NF-κB activation and expression of multiple inflammatory signals, such as tumor necrosis factor-α (TNF-α), cytokine-inducible neutrophil chemoattractant (CINC), and intercellular adhesion molecule-1(ICAM-1) by ELISA methods. Serum alanine aminotransferase (ALT), intrahepatic myeloperoxidase (MPO)/WBC (a measure of neutrophil accumulation) and Mac-1 expression (a measure of circulating neutrophil activity) were also evaluated.RESULTS: PDTC decreased NF-κB activation induced by prolonged cold preservation in a dose dependent manner (from 20 mmol/L to 60 mmol/L), diminished TNF-α, CINC,ICAM-1 proteins in the grafts, and reduced the expression f increases in plasma TNF-α levels induced by prolonged old preservation. Neutrophilic inflammation of the graft was significantly suppressed after preservation with PDTC (P&lt;0.05). The total neutrophil accumulation in PDTC (40 mmol/L) group (7.04&#177;0.97) was markedly reduced compared to control group (14.07&#177;1.31) (P&lt;0.05). Mac-1 expression was significantly reduced in PDTC (40 retool/L) group (181&#177;11.3%) compared with the control group (281&#177;13.2%) (P&lt;0.05) at 6 h after reperfusion. Furthermore,PDTC inhibited the increased serum ALT levels after liver transplantation.CONCLUSION: PDTC can inhibit B NF-κB activation and expression of the inflammatory mediators, which are associated with improved graft viability via inhibiting intrahepatic neutrophilic inflammation. Our study suggests that a therapeutic strategy directed at inhibition of NF-κB activation in the transplanted liver might be effective in reducing intrahepatic neutrophilic inflammation, and would be beneficial to cold preserved grafts,     

基质细胞衍生因子/趋化因子受体4轴参与胰腺癌细胞侵袭的体外实验研究

Objective To investigate the expression of chemokine receptor (CXCR)4 in human pancreatic cancer cell lines,and its association with proliferation,adhesion and invasion of pancreatic cancer cells.Methods The CXCR4 mRNA and protein in three pancreatic cancer cell lines were detected by RT-PCR and Western blotting,respectively.Confocal microscopy was used to detect the fluorescence intensity induced by SDF-lα in AsPC-1 cells.MTT test was performed to study the proliferation of pancreatic cancer cells.The invasive ability of pancreatic cell lines was determined by transwell invasion assay kit and the adhesive ability was detected by cell adhesive test in vitro.Results There were expressions of CXCR4 mRNA and protein in different extent in three pancreatic cancer cell lines.The strong expression was seen in AsPC-1 cell line,but weak expression in SW1990 cell line.The CXCR4 was functional expressed on AsPC-1 ceils.SDF-1α improved the proliferation,adhesion and invasion of three pancreatic cancer cell lines,especially in AsPC-1 cell line,while the proliferation in SW1990 cell line was weak.But all above effects of the SDF-1α could be inhibited by AMD3100.Conclasions CXCR4 mRNA and protein were expressed in pancreatic cancer cell lines.The efficacy that SDF-1 can increase the invasive ability of pancreatic cancer ceils through SDF-1/CXCR4 axis is closely related to the expression of CXCR4.     

基质细胞衍生因子/趋化因子受体4轴参与胰腺癌细胞侵袭的体外实验研究

Objective To investigate the expression of chemokine receptor (CXCR)4 in human pancreatic cancer cell lines,and its association with proliferation,adhesion and invasion of pancreatic cancer cells.Methods The CXCR4 mRNA and protein in three pancreatic cancer cell lines were detected by RT-PCR and Western blotting,respectively.Confocal microscopy was used to detect the fluorescence intensity induced by SDF-lα in AsPC-1 cells.MTT test was performed to study the proliferation of pancreatic cancer cells.The invasive ability of pancreatic cell lines was determined by transwell invasion assay kit and the adhesive ability was detected by cell adhesive test in vitro.Results There were expressions of CXCR4 mRNA and protein in different extent in three pancreatic cancer cell lines.The strong expression was seen in AsPC-1 cell line,but weak expression in SW1990 cell line.The CXCR4 was functional expressed on AsPC-1 ceils.SDF-1α improved the proliferation,adhesion and invasion of three pancreatic cancer cell lines,especially in AsPC-1 cell line,while the proliferation in SW1990 cell line was weak.But all above effects of the SDF-1α could be inhibited by AMD3100.Conclasions CXCR4 mRNA and protein were expressed in pancreatic cancer cell lines.The efficacy that SDF-1 can increase the invasive ability of pancreatic cancer ceils through SDF-1/CXCR4 axis is closely related to the expression of CXCR4.     

基质细胞衍生因子/趋化因子受体4轴参与胰腺癌细胞侵袭的体外实验研究

Objective To investigate the expression of chemokine receptor (CXCR)4 in human pancreatic cancer cell lines,and its association with proliferation,adhesion and invasion of pancreatic cancer cells.Methods The CXCR4 mRNA and protein in three pancreatic cancer cell lines were detected by RT-PCR and Western blotting,respectively.Confocal microscopy was used to detect the fluorescence intensity induced by SDF-lα in AsPC-1 cells.MTT test was performed to study the proliferation of pancreatic cancer cells.The invasive ability of pancreatic cell lines was determined by transwell invasion assay kit and the adhesive ability was detected by cell adhesive test in vitro.Results There were expressions of CXCR4 mRNA and protein in different extent in three pancreatic cancer cell lines.The strong expression was seen in AsPC-1 cell line,but weak expression in SW1990 cell line.The CXCR4 was functional expressed on AsPC-1 ceils.SDF-1α improved the proliferation,adhesion and invasion of three pancreatic cancer cell lines,especially in AsPC-1 cell line,while the proliferation in SW1990 cell line was weak.But all above effects of the SDF-1α could be inhibited by AMD3100.Conclasions CXCR4 mRNA and protein were expressed in pancreatic cancer cell lines.The efficacy that SDF-1 can increase the invasive ability of pancreatic cancer ceils through SDF-1/CXCR4 axis is closely related to the expression of CXCR4.     

基质细胞衍生因子/趋化因子受体4轴参与胰腺癌细胞侵袭的体外实验研究

Objective To investigate the expression of chemokine receptor (CXCR)4 in human pancreatic cancer cell lines,and its association with proliferation,adhesion and invasion of pancreatic cancer cells.Methods The CXCR4 mRNA and protein in three pancreatic cancer cell lines were detected by RT-PCR and Western blotting,respectively.Confocal microscopy was used to detect the fluorescence intensity induced by SDF-lα in AsPC-1 cells.MTT test was performed to study the proliferation of pancreatic cancer cells.The invasive ability of pancreatic cell lines was determined by transwell invasion assay kit and the adhesive ability was detected by cell adhesive test in vitro.Results There were expressions of CXCR4 mRNA and protein in different extent in three pancreatic cancer cell lines.The strong expression was seen in AsPC-1 cell line,but weak expression in SW1990 cell line.The CXCR4 was functional expressed on AsPC-1 ceils.SDF-1α improved the proliferation,adhesion and invasion of three pancreatic cancer cell lines,especially in AsPC-1 cell line,while the proliferation in SW1990 cell line was weak.But all above effects of the SDF-1α could be inhibited by AMD3100.Conclasions CXCR4 mRNA and protein were expressed in pancreatic cancer cell lines.The efficacy that SDF-1 can increase the invasive ability of pancreatic cancer ceils through SDF-1/CXCR4 axis is closely related to the expression of CXCR4.