AIM:To investigate the activation and inactivation of nuclear factor kappa B(NF-κB)when tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)is applied to induce the apoptosis of androgen-independent prostate cancer cell line PC-3M.METHODS:After the treatment of TRAIL or LPS at different doses,we tested the nuclear translocation of NF-κB by cell immunohistochemical staining and electrophoretie mobility shift assay(EMSA),and evaluated the level of IκB by RT-PCR under pyrrolidine dithiocarbamate(PDTC)treatment.RESULTS:EMSA and cell immunohistochemical analysis showed that the translocation of NF-κB was significantly activated when PC-3M cells were treated with TRAIL or LPS(P＜0.05).The pretreatment of PDTC upregulated the expression of IκB and blocked the nuclear translocation of NF-κB.CONCLUSION:TRAIL remarkably stimulates the activation of nuclear NF-κB in androgen-independent prostate cancer cells.On the other hand,the translocation of NF-κB can be significantly and efficiently inhibited in PC-3M cells by pretreatment with PDTC.The increased expression of IκB might be a clue for this inhibition,which means the possible way to enhance the effect of TRAIL in the apoptosis of prostate cancer cells.

AIM:To evaluate the ability of dendritic cells (DCs) loaded with tumor lysate to initiate cell mediated immune responses by stimulating naive T cells, and the efficiency of activated T cells to kill autologous tumor cells in vitro. METHODS: The peripheral blood lymphocytes and monocytes were obtained from the advanced renal cell carcinoma patient by eonglutination method. The immature dendritic cells were generated in the presence of interleukin -4(IL-4) and granulocyte/macrophage colony-stimulating factor (GM-CSF) from monocytes of healthy individuals.These cells were pulsed with tumor lysate or not. Induction of tumor-specific cytotoxic T lymphocytes(CTLs) response by mature dendritic cells (mDCs) was evaluated by the CD95(Fas) expression assay through FCM and the cytotoxic assay a gninst autolognns human tumor cells. RESULTS: Human immature dendritic cells and T cells obtained from healthy donors were stimulated with tumor- pulsed dendritic cells. The immature dendritic cells were applied to the cytotoxicity assay a gainst target autologons tumor cells. The CD95 (Fas) expression, IFN-γ, and TNF -α secreted by the CTLs in tumor lysate-plused DC group were higher than those of other groups. The capacity of the CTLs to kill autolognns tumor cells was significantly different(P<0. 05). Antigen-specific DCs vaccine can induce T cells activation and proliferation, thus we can obtain higher proportion of tumor specific cytotoxic T cells(CTLs), and enhance the CTLs to secret IFN-γ and TNF-α. CONCLUSION: Our results indicate that monocyte-derived human dendritic cells pulsed with tumor lysate could in duce the specific antitumor effect against autologons tumors. This in vitro model offers a new and simple approach to the development of DC + CTL - based immunotherapy.

AIM: To verify the protection of astragalus polysaecharide (APS) on H2O2 - stressed skin fibro-blasts. METHODS: A model of acute H2O2 stress in primary skin fibroblast was used at concentration of 0.5 mmol/L by 30 rain incubation. Dose responses of APS on cell survival was measured by MTI, cell death was evaluated by DAPI, and effect of APS on mitochondria, mitochondrial membrane potential and lysesome stabilization were measured by confocal microscopy. RESULTS: APS improved cell survival in a dose -dependent manner, starting at 0. 5 mg/L and with a maximum at 1 mg/L. Moreover, APS inhibited mitochondrial membrane potential collapse, protected mitechondrial morphology and stablized lysosomal membrane. CONCLUSION: The results suggest the existence, at the mitochondria-lysosome level, of a new pathway of apoptotic regulation by APS. This might constitute a new therapeutic target where oxidative stress and lysesomal impairment are involved.

AIM:To investigate the effect of non-mitogenic human acidic fibroblast growth factor(nm-haFGF)on renal ischemia-reperfusion injury in rats.METHODS:Rat renal ischemia-reperfusion(I/R)injury was produced by removing the left kidney and subsequently clamping the right renal artery for 60 min followed by reperfusion for 24 h.5 min after reperfusion.different doses of nm-haFGF and haFGF(as positive control)were injected by lingual vein.24 h later,the samples of blood,urine and kidney were collected and the contents of malondialdehyde(MDA),blood urea nitrogen(BUN),creatinine(Cr)and superoxide dismutase(SOD)activity were detected.Histopathological changes were also observed.RESULTS:In the serum,SOD activity of all the nm-haFGF groups and the haFGF group increased significantly while the content of MDA decreased dramatically compared with the model group;The content of BUN and Cr aland haFGF group rose significantly compared with the model group,while MDA decreased dramatically.Histological examination showed that nm-haFGF markedly attenuates the renal edema,brush border's defluvium and cell necrosis induced by ischemia-reperfusion.CONCLUSION:nm-haFDF could resist the renal injury induced by ischemia-reperfusion in rats.

AIM:To detect and compare the longitudinal mitral annulus diastolic velocity and time interval changes by pulsed Doppler tissue imaging(DTI)in patients with angina pectoris(AP)and myocardial infarction(MI),and to explore the value of mitral annulus diastolic velocities and time intervals for evaluation of global left ventricular diastolic dysfunction.METHODS:Fifty patients with established coronary artery disease were divided into AP group(16 cases)and MI group(34 cases).Sixteen age-matched healthy individuals served as the control group.The septum,lateral,anterior and inferior walls of the mitral annulus were displayed,and selected for DTI spectrum sampling.Peak early and late diastolic velocities and their ratio,time to the onset and peak of the early diastolic wave,and regional isovolumic relaxation time were measured,and the average values of the four mitral annular sites were calculated and presented as Em,Am,Em/Am,QEm,TEm and IVRTm,respectively.RESULTS:Compared with the control group,Em and Em/Am were significantly lower in both the AP and the MI groups(P＜0.01).Em was even lower in the MI group than that in the AP group(P＜0.01).QEm,TEm and IVRTm were significantly longer in the AP and the MI groups than those in control group(P＜0.01 or P＜0.05).IVRTm was even longer in the MI group than that in AP group(P＜0.01).IVRTm had significantly negative correlation with Em(r=-0.64,P＜0.01).CONCLUSION:Em,Em/Am,QEm,TEm and IVRTm as measured by pulsed DTI may be promising indexes for quantitative assessment of global left ventricular diastolic dysfunction in patients with coronary artery disease.Em and IVRTm may indicate the severity of ischemic myocardial damage.

AIM: Direct exposure of cells to reactive oxygen species can induce apoptosis. In this study we investigate how oxidative stress induces cell death in HepG2 cells and characterize the molecular events involved. METHODS: Oxidative stress was created by exposing HepG2 cells to 2 mmol/L H2O2. Apoptosis was determined by analysis of DNA fragmentation by agarose gel electorphoresis. The mitochondrial membrane potential was analyzed using DePsipher fluorescent staining and the expression of cytochrome c in the cytosolic fraction was measured by Western blotting analysis.The caspase activity was detected using fluorometric assay kit by a fluorescence microplate reader. RESULTS: When HepG2 cells were treated with 2 mmol/L H2O2, the cells displayed DNA fragmentation, a typical feature of apoptosis, after 12 h. The mitochondrial membrane potential appeared different in two group of cells. H2O2 -treated cells appeared green fluorescence as early as 4 h, which represents de - energized mitochondria, the untreated cells appeared red fluorescence,a feature of mitochondria with intact membrane potential. In treated cells, the expression of cytochrome c increased and accumulated in cytosolic fraction with treatment time, caspase - 3 activity increased by 6.7 - fold ( P ＜ 0.01 ) at 8 h and caspase -9 activity increased by 3.6 - fold (P ＜ 0.01 ) at 12 h, respectively, however, the activity of caspase - 8 remained unchanged. CONCLUSION: These findings suggest that oxidative stress can induce apoptotic cell death in HepG2 cells, and the mechanism is related to mitochondrial pathway, which activates caspase -9 and- 3, but not caspase -8.

AIM: To investigate the apoptosis of implanted tumor of primary human gastric cancer cells in nude mice induced by genistein and the relation between this apoptosis and expression of bcl - 2 and bax. METHODS: Establishing a transplanted tumor model by injecting human primary gastric cancer cells into subcutaneous tissue of nude mice. The different doses of genistein (0.5mg/kg, 1mg/kg and 1.5 mg/kg ) were directly injected beside tumor body respectively,for six times at an interval of two days. Then changes of tumor volume were measured continuously and tumor inhibition rate of each group was calculated. We observed the morphologic alteration by electron microscope, measured the apoptotic rate by TUNEL staining method, detected the expression of apoptosis - regulated gene bcl - 2 and bax by immunohistochemical staining and RT- PCR. RESULTS: Genistein could significantly inhibit carcinoma growth when it was injected near the carcinoma. Genistein induced implanted tumors cells to undergo apoptosis with apoptotic characteristics by transmission electron microscope. The apoptosis index of above three groups was increased progressively. Positive rate of Bcl - 2 protein of above three groups was decreased progressively and positive rate of Bax protein of above three groups was increased progressively by immunohistochemical staining. The density of bcl -2 mRNA decreased progressively and the density of bax mRNA increased progressively with elongation of time by RT - PCR. CONCLUSION: Genistein is able to induce the apoptosis of transplanted tumor cells. This apoptosis may be mediated by down - regulating bcl - 2 and up - regulating bax mRNA and its protein.

AIM: To investigate whether RNA interference (RNAi) induced by small interference RNA (siRNA) could suppress Polo- like kinase- 1 (Plk 1 ) expression and its effects in A549 cells. METHODS: A recombinant plasmid containing siRNA targeting Plk1 ( psiRNA - hH1 - Plk1 ) was transfected into A549 cells with Lipofectamine 2000.Expressions of Plk1, cyclin B1 and p53 protein were detected by Western blotting. Cell proliferation was evaluated by direct cell counting, while cell cycle and apoptosis were examined by flow cytometry, and expression of α - tubulin was detected by immunofluorescence. RESULTS: The results demonstrated that sequence specific siRNA targeting Plk1 was capable of suppressing Plk1 expression, and reflecting in lower kinase activity in A549 cells. The level of Plk1 protein was reduced by at least 70％ after 48 h of psiRNA - hH1 - Plk1 treatment relative to controls. Expressions of cyclin B1 and p53 were increased greatly after Plk1 depletion, and cells showed absence of microtubule polymerization and spindle abnormalities in staining for α -tubulin. Growth inhibition, G2/M arrest and apoptosis were observed in psiRNA -hH1 -Plk1 transfected group. CONCLUSION: All these data suggest that siRNA targeted against human Plk1 may be a valuable tool in cancer therapy.

AIM:To research the characteristics of ventricular electrophysiology in right ventricular rapid pacing-induced congestive heart failure (CHF) dogs. METHODS:Dogs (n = 16) were randomly divided into 2 groups:the control (n = 7) and the CHF group (n = 9) induced by rapid right ventricular pacing at 240 pulse·min-1 for 4 to 5 weeks. The electrophysiologic parameters were evaluated by the technique of standard electric stimulation and monophasic action potential (MAP) recording. RESULTS:(1) Ventricular effective refractory period (VERP), ventricular MAP duration (MAPD90), ventricular late repolarization duration (VLRD) and intra- ventricular conduction time (IVCT) were prolonged by 26％ (P ＜ 0. 01), 43％ (P ＜ 0. 01), 318％ (P ＜ 0. 05), and 19％ (P ＜ 0. 01), respectively in CHF group. (2)The ratio of VERP to MAPD90(VERP/MAPD90) was decreased by 13％ (P＜0.05) in CHF group. (3) The dispersion of ventricular recovery time (VRT - D) was increased by 185％ (P ＜0. 01) in CHF group. (4) The ventricular fibrillation threshold (VFT) was decreased by 48％ (P ＜ 0. 01) in CHF group. CONCLUSION:The abnormal electrophysiological changes in the CHF condition may be contributing factors of lethal ventricular arrhythmias and sudden cardiac deaths in CHF.

AIM: To investigate the influence of mesenchymal stem cells (MSCs) expressing indoleamine 2, 3-dioxygenase (IDO) activity on the allogeneic T-lymphocyte responses.METHODS: MSCs were isolated and cultured from human bone marrow. Selected surface markers of MSCs were detected by flow cytometry and their morphologic characteristics were determined by microscopy. The pluripotentiality of MSCs was also studied. IDO mRNA and IDO protein expressions in MSCs induced by γ-interferon (IFN-γ) at the concentration of 2×105 U/L were detected. MSCs induced by IFN-γ at the concentration of 2×105 U/L were plated in dishes and then mixed lymphocyte reaction (MLR) cultures were set up. T-lymphocytes proliferation was determined by MTT assays and IDO activity was measured by high-performance liquid chromatography (HPLC). RESULTS: IFN-γ could stimulate IDO mRNA and IDO protein expressions in MSCs. IDO enzyme activity in induced MSCs inhibited T-lymphocyte proliferation of MLR cultures. CONCLUSION: MSCs could suppress T-lymphocyte responses via IDO enzyme activity in vitro.