Inhibition of PMA-induced endothelial cell activation and adhesion by over-expression of domain negative IκBα protein

AIM: NE-κB, regulate the expression of cytokine-inducible genes involving immune and inflammatory responses, will be potential therapy approach for allograft from rejection.In this study, we use pCMV-IκBαM vector to inhibit NE-κB activation and investigate the effect of pCMV-IκBαM in inhibition of T cells adhesion to endothelial cells.METHODS: The NF-κB activity was detected with pNF-κB reporter gene and electrophoretic mobility shift assay.Expression of cell surface molecules was detected by RT-PCR and flow cytometer. The cell-cell adhesion assay was performed to determine the effect of pCMV-I~BczMin in hibition of T cells adhesion to endothelial cells.RESULTS: We could find that NF-~B activity is inhibited by over-expression of non-degraded IκBα protein.Expression of adhesion molecules like ICAM-1, VCAM-1,and P-selectin as well as cell-cell adhesion were inhibited significantly by transfection of the pCMV-IκBαM vector.CONCLUSION: Our results indicate that the pCMV-IκBαM, which inhibit the activity of NF-κB through over-expression of non-degraded IκBα protein, can be used for gene therapy in diseases involving NF-κB activation abnormally like organ transplantation via inhibiting cell adhesion.     

Hepatic preconditioning of doxorubicin in stop-flow chemotherapy:NF-κB/IκB-α pathway and expression of HSP72

AIM: To provide hepatic protection through administration of doxorubicin before stop-flow chemotherapy (SFC) and to investigate the expression of heat shock protein 72 (HSP72) and role of nuclear factor kappa B (NF-κB) in this effect. METHODS: The hepatic preconditioning of doxorubicin was established in a porcine model by injection of doxorubicin (1 mg/kg) before SFC. The experimental animals were randomized into two groups: groups receiving doxorubicin (DOX) and normal saline (NS). Serial serum and tissue samples were taken from both groups to evaluate the protection of doxorubicin. Western blot and immunoprecipitation were applied to detect the expression of HSP72, NF-κB p65 protein, inhibitor κB-α (IκB-α) and phosphorylated IκB-α as well. The expression of tumor necrosis factor α (TNF-α) was estimated by semiquantitative RT-PCR. And the extent of the hepatic injury was estimated with the level of serum aminotransferases. RESULTS: An abundance production of HSP72 in porcine liver was observed after 24 h of intravenous administration of doxorubicin, but without any change in the expression of NF-κB p65 subunit in cytoplasm. NF-κB p65 subunit accumulated in nuclei at the end of SFC and reached its highest level at 30 min after the restoration of the abdominal circulation and decreased gradually during the 6 h after SFC in NS group, while there was little change in DOX group. There was also a slight decrease of IκB-α at 30 min after the restoration of the abdominal circulation in NS group accompanying with the appearance of phosphorylated IκB-α. The expression of TNF-α was significantly higher in NS group than that in DOX group (average 1.40±0.17 vs 0.62±0.22, P<0.01) at serial time points after SFC. Serum ALT and AST levels of NS group were higher after 24 h than those of DOX group (93.2±7.8 IU/L vs 53.3±13.9 IU/L, 217.0±29.4 IU/L vs 155.0±15.6 IU/L for ALT and AST respectively, P<0.05) and after 48 h than those of DOX group (66.6±18.1 IU/L vs 43.3±16.7 IU/L, 174.4±21.3 IU/L vs 125.7±10.5 IU/L for ALT and AST respectively, P<0.05). CONCLUSION: Doxorubicin renders the liver to be tolerant to the hepatic influence in SFC in a porcine model through the NF-κB/IκB-αpathway with the expression of HSP72.     

Effect of mutant p27(kipl) gene on human cholangiocarcinoma cell line, QBC(939)

AIM：To investigate the effects of exogenously mutated p27^kip1 （p27） on proliferation and apoptosis of human cholangiocarcinoma cell line, QBC939 in vivo.METHODS： Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line. Expression of p27 was detected by RT-PCR. Western blot. Cell growth, morphological change, cell cycle, apoptosis and cloning formation were determined by MTT assay and flow cytometry.RESULTS： The expression of p27 protein and mRNA was increased signifi cantly in QBC939 cell line transfected with Ad-p27mt. The transfer of Ad-p27mt could signifi cantly inhibit the growth of QBC939 cells, decrease the cloning formation rate and induce apoptosis. p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Ad-p27mt.CONCLUSION： p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis. Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.     

Effects of interleukin-10 on activation and apoptosis of hepatic stellate cells in fibrotic rat liver

AIM:To study the effects of interleukin-10(IL-10)on the expression of α-smooth muscle actin(α-SMA),nuclear factor-κB(NF-κB)and Fas/Fas ligand(FasL)inhepatic stellate cells of experimental rats with hepaticfibrosis.METHODS:Sixty clean SD rats were randomly dividedinto control group(group N),liver fibrotic group(groupC)and IL-10 treatment group(group I).Control groupreceived intraperitoneal injection of saline(2ml·kg~(-1)),twicea week.Fibrotic group was injected intraperitoneallywith 50% carbon tetrachloride(CCl_4)(2 ml·kg~(-1)),twicea week.IL-10 treatment group was given IL-10 at adose of 4 μg·kg~(-1)20 minutes before CCl_4 administrationfrom the third week.Hepatic stellate cells(HSCs)wereisolated from these rats at the seventh and eleventhweeks during the course of liver fibrosis,respectively.The expression of α-SMA and NF-κB in HSCs wasmeasured by S-P immunohistochemistry.The expressionof Fas and FasL mRNA was measured by RT-PCR.Furthermore,liver tissues were harvested from threegroups at the same time.RESULTS:The CCl_4- induced experimental rat hepaticfibrosis model was established successfully.The purityof extracted hepatic stellate cells was about 95% andthe yield of hepatic stellate cells was 1.2-2.3×10~6/g livertissue averagely.The positive expression of α-SMA andNF-κB was 36.5% and 28.5% respectively in group N.The positive levels of α-SMA and NF-κB were increasedsignificantly in group C compared to group N(P<0.01).The positive signals decreased significantly(P<0.05)ingroup I.In the 11~(th)week,the HSCs of group I becameround with visible pyknotic nuclei.The expression ofNF-μB in group C was significantly increased in a time-dependentmanner(P<0.01),but there was no difference in the α-SMA expression(P>0.05).The mRNA of Fasand FasL in group C was significantly increased in a time-dependent manner compared to that in control group.After treated with IL-10,the expression level of Fas andFasL was higher in group I than in group C.CONCLUSION:The positive expression of α-SMA andNF-κB in hepatic stellate cells is decreased by ectogenicIL-10 in liver fibrosis induced by CCl_4.The expression ofFas and FasL is increased in the course of liver fibrosis,and is further increased by IL-10.IL-10 could inhibitthe activation of HSCs and cause apoptosis of activatedHSCs.     

急性冠状动脉综合征患者外周血单个核细胞核因子-κB蛋白表达水平与冠状动脉病变特征的关系

Objective To explore the correlation between expression of nuclear factor kappa B (NF-kappa B) in peripheral blood monouclear cells(PBMC) and the severity of coronary artery lesion in patients with acute coronary syndrome(ACS). Methods The 81 patients were diagnosed with coronary heart disease, and all of them underwent immediate or selective coronary angiography(CAG) The nuclear protein level of activated NF-B was detected by Western blot and semi-quantity image analysis was done. Coronary angiograms were scored according to Gensini integration. The relationship between NF-κB and Gensini score was analyzed. Results The protein expression of NF-κB in PBMC in ACS group was significantly higher than that in stable angina group and control group (0.85±0.18 vs. O.75±0.21 and 0.71±0.23, F=3.72, P<0.05). The protein expression of NF-κB was not correlated with Gensini score(r=0.07, P>0.05). Conclusions The ACS patients have the activation of NF-κB in PBMC. The protein expression of NF-κB is not correlated with Gensini score, which does not suggest an implication of the severity of coronary artery.     

Nuclear factor-KB decoy oligodeoxynucleotides attenuates ischemia/reperfusion injury in rat liver graft

AIM: To evaluate the protective effect of NF-κB decoy oligodeoxynucleotides (ODNs) on ischemia/reperfusion (I/R) injury in rat liver graft. METHODS: Orthotopic syngeneic rat liver transplantation was performed with 3 h of cold preservation of liver graft in University of Wisconsin solution containing phosphorothioated double-stranded NF-κB decoy ODNs or scrambled ODNs. NF-κB decoy ODNs or scrambled ODNs were injected intravenously into donor and recipient rats 6 and 1 h before operation, respectively. Recipients were killed 0 to 16 h after liver graft reperfusion. NF-κB activity in the liver graft was analyzed by electrophoretic mobility shift assay (EMSA). Hepatic mRNA expression of TNF-α, IFN-γ and intercellular adhesion molecule-1 (ICAM-1) were determined by semiquantitative RT-PCR. Serum levels of TNF-α and IFN-γ were measured by enzyme-linked immunosorbent assays (ELISA). Serum level of alanine transaminase (ALT) was measured using a diagnostic kit. Liver graft myeloperoxidase (MPO) content was assessed. RESULTS: NF-κB activation in liver graft was induced in a time-dependent manner, and NF-κB remained activated for 16 h after graft reperfusion. NF-κB activation in liver graft was significant at 2 to 8 h and slightly decreased at 16 h after graft reperfusion. Administration of NF-κB decoy ODNs significantly suppressed NF-κB activation as well as mRNA expression of TNF-α, IFN-γ and ICAM-1 in the liver graft. The hepatic NF-κB DNA binding activity [presented as integral optical density (IOD) value] in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (2.16±0.78 vs 36.78 ±6.35 and 3.06±0.84 vs 47.62± 8.71 for IOD value after 4 and 8 h of reperfusion, respectively, P<0.001). The hepatic mRNA expression level of TNF-α, IFN-γ and ICAM-1 [presented as percent of p-actin mRNA (%)] in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (8.31 ±3.48 vs 46.37±10.65 and 7.46± 3.72 vs 74.82±12.25 for hepatic TNF-a mRNA, 5.58±2.16 vs 50.46±9.35 and 6.47±2.53 vs 69.72±13.41 for hepatic IFN-y mRNA, 6.79 ±2.83 vs 46.23±8.74 and 5.28±2.46 vs 67.44±10.12 for hepatic ICAM-1 mRNA expression after 4 and 8 h of reperfusion, respectively, P<0.001). Administration of NF-κB decoy ODNs almost completely abolished the increase of serum level of TNF-α and IFN-γ induced by hepatic ischemia/reperfusion, the serum level (pg/mL) of TNF-α and IFN-γ in the NF-kB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (42.7±13.6 vs 176.7±15.8 and 48.4±15.1 vs 216.8±17.6 for TNF-α level, 31.5±12.1 vs 102.1±14.5 and 40.2±13.5 vs 118.6±16.7 for IFN-γ level after 4 and 8 h of reperfusion, respectively, P<0.001). Liver graft neutrophil recruitment indicated by MPO content and hepatocellular injury indicated by serum ALT level were significantly reduced by NF-κB decoy ODNs, the hepatic MPO content (A655) and serum ALT level (IU/L) in the NF-κB decoy ODNs treatment group rat was significantly lower than that of the I/R group rat (0.17±0.07 vs 1.12 ±0.25 and 0.46±0.17 vs 1.46±0.32 for hepatic MPO content, 71.7±33.2 vs 286.1±49.6 and 84.3±39.7 vs 467.8±62.3 for ALT level after 4 and 8 h of reperfusion, respectively, P< 0.001). CONCLUSION: The data suggest that NF-κB decoy ODNs protects against I/R injury in liver graft by suppressing NF-κB activation and subsequent expression of proinflammatory mediators.     

WWOX基因转染对胆管癌细胞增殖、凋亡及侵袭的影响

目的:探讨WWOX基因转染胆管癌细胞株QBC939后对其增殖、凋亡与侵袭性的影响.方法:用脂质体转染法将WWOX重组真核表达质粒转染QBC939细胞,建立稳定表达WWOX基因的细胞株.将其分为以下3组:QBC939组,QBC939/con组和QBC939/WWOX组.荧光定量RT-PCR和Western blot法检测...     

Survivin对人胆管癌细胞凋亡相关信号通路的作用机制

目的:探讨Survivin基因对人胆管癌细胞凋亡信号通路的调节机制.方法:构建针对Survivin基因的siRNA和对照siRNA,分别转染QBC939人胆管癌细胞,Western blot检测siRNA对细胞Survivin的干扰效果.继而分别用流式细胞仪,激酶活性测定和Westernblot检测不同Survivin表达状态下,QBC939细胞的凋亡状态,caspase-3的活性和caspase-3,caspase-9及procaspase-9凋亡信号分子的表达.结果:siRNA-Survivin显著抑制Survivin在QBC939细胞的表达(P<0.05).Survivin表达抑制后,QBC939细胞凋亡明显增加(18.9%±2.3%,P<0.05),caspase-3活性显著升高(0.83±0.15,P<0.01),caspase-3和caspase-9表达明显上调(P<0.05),而procaspase-9表达降低(P<0.05).未转染和转染对照siRNA的QBC939细胞上述变化无显著性差异(P>0.05).结论:Survivin基因通过促进procaspase-9的活化以阻止caspase-3和caspase-9的激活从而抑制胆管癌细胞的凋亡.     

Effect of mutant p27kip1 gene on human cholangiocarcinoma cell line, QBC939

AIM:To investigate the effects of exogenously mutated p27kip1 (p27) on proliferation and apoptosis of human cholangiocarcinoma cell line,QBC939 in vivo.METHODS:Adenviral vectors were used to transfect mutated p27 cDNA into human QBC939 cell line.Expression of p27 was detected by RT-PCR.Western blot.Cell growth,morphological change,cell cycle,apoptosis and cloning formation were determined by MTT assay and flow cytometry.RESULTS:The expression of p27 protein and mRNA was increased significantly in QBC939 cell line transfected with Ad-p27mt.The transfer of Adp27mt could significantly inhibit the growth of QBC939cells,decrease the cloning formation rate and induce apoptosis,p27 over expression caused cell cycle arrest at G0/G1 phase 72 h after infection with Adp27mt.CONCLUSION:p27 may cause cell cycle arrest at G0/G1 phase and subsequently lead to apoptosis.Recombinant adenovirus expressing mutant p27 may be potentially useful in gene therapy for cholangiocarcinoma.     

Construction of HCV—core gene vector and its expression in cholangiocarcinoma

AIM: To establish an experimental model for exploring the role of hepatitis C virus (HCV) in the development of cholangiocarcinoma. METHODS: Recombinant plasmid of HCV-core gene was constructed with molecular cloning technique and transfected into QBC939 cells with lipofection.After it was selected with G418, resistant colonies were obtained. The colonies were analysed by immunocytochemistry and Western blotting.The morphology was observed under transmission electron microscope(TEM) and microscope. RESULTS: The recombinant plasmid was proved to carry the target gene by PCR and restriction enzymed mapping. Moreover, it could express HCV-C protein efficiently in QBC939 cells. The HCV-like particles were found in the cytoplasm by electron microscope, which were spherical with a diameter of 50 nm-80 nm possessing outer membrane.The transfected cells had lower differentiation and higher malignant degree under microscope. CONCLUSION: Because HCV-core gene could express steadily in cholangiocarcinoma cells,the transfected tumor cells(QBC939-HCVC) could be used to study the effect of HCV in the development of cholangiocarcinoma.     

阿托伐他汀对人胆管癌QBC939细胞系侵袭的影响

目的探讨阿托伐他汀(ATV)对人胆管癌QBC939细胞系侵袭的影响及其可能作用机制。方法应用细胞培养技术培养人胆管癌QBC939细胞,经不同浓度的ATV处理后,以Matrigel侵袭实验和半定量RT-PCR检测ATV对QBC939细胞内Rho C mRNA表达的影响情况。结果 Matrigel侵袭实验显示,经10μmol/L、25μmol/L、50μmol/L干预48 h后的QBC939细胞,随着浓度的增加,其体外侵袭能力明显减弱(P0.01);半定量RT-PCR测定显示,ATV作用48 h后,QBC939细胞中Rho C的表达改变并不明显。结论 ATV可减弱人胆管癌QBC939细胞体外侵袭能力。     

Fas counterattack in cholangiocarcinoma: a mechanism for immune evasion in human hilar cholangiocarcinomas

AIM:To investigate FasL expression in hilarcholangiocarcinoma tissues and culturedcholangiocarcinoma cells,and to assess its ability to induceapoptosis.METHODS:We studied the expression of FasL by humanhilar cholangiocaroinomas tissues byimmunohistochemistry,and the QBC939 cholangiocarcinomacell line by FIT-PCR,immunohistochemistry,and WesternBlot.TUNEL and flow cytometry were used to detectapoptotic cells.RESULTS:Prevalent expression of FasL was detected in 39resected hilar cholangiocarcinoma tissues.TUNEL slainingdisclosed a high level of call death among lymphocytesinfiltrating FasL positive areas of tumor.FasL mRNA andprotein expressions in cholangiocarcinoma cells couldinduce Jurkat cells.OONCLUSION:Hilar cholangiocarcinomas may eludeimmunological surveiliance by inducing,via Fas/FasLsystem,the apoptosis of activated lymphocytes.