Objective:To investigate the availability for rhIL-18 in the in vitro culture system to stimulate PBMHCs,with inducing cytotoxicities against tumor cells and to analyze the influencing factors for the effects.Methods:The NK cells,T cells and dendritic cells were separated from fresh PBMNCs by using the Stem Sep TM immunomagnetic beads.Cell phenotypes of the purified cell populations were identified with FCM technique.The cell co-culture system was established as follows.The PBMNCs or the cell preparations deleting the definite cell subset with immunoscreening were co-cultivated with mitotic-inactivated tumor cells in the presence of rhIL-18.Cytotoxicities of the various effector cell preparations to a series of tumor cell lines were examined by the isotope releasing assay.Results:In the in vitro cell co-culture system,rhIL-18 rapidly induces activation of the cytolytic responses against various tumor cell lines mediated by PBMNCs.The rapid induction within 24 h of culture was dependent on the dosage of rhIL-18,with the optimal dose of 100 ng/ml of the cytokine.The activated cytotoxicities were abrogated by deleting NK cells prior to the cell co-culture but did not vary when either T cells or DCs were removed.The cytotoxic responses were shown as a pattern of broad-spectrum to the target cells used and were not blocked by anti-MHC moAbs.Conclusion:NK cells were responsible for the rapid induction of cytotoxicities against tumor cells by rhIL-18.

Aim To study gyrA and parC mutations of clinical Pseudomonas aeruginosa strains. Methods MIC values of 55 clinical P.aeruginosa isolates were determined by agar dilution test and 1 sensitive strain and 8 resistant strains were selected with standard sensitive strain ATCC27853 as control, the quinolone determining region (QRDR) of the gyrA and parC genes were amplified by PCR, the lengths of PCR products were 351 bp and 397 bp. The gyrA PCR products(351 bp) were digested with enzyme sacⅡ. The gyrA and parC gene were sequenced. Results In this study, gyrA genes of all resistant strains had an ACC to ATC mutation in codon 83, leading to the amino acid substitution of an isoleucine for a threonine, and three high level resistant strains also showed a GAC to GGC mutation in codon 87, leading to the substitution of a glycine for an aspartic acid. In addition, four resistant strains also had an TCG to TTG mutation in codon 87 of parC gene, leading to the amino acid substitution of a serine for a leucine. The strains with both gyrA and parC mutations were two to sixteen times more resistant than the strains which had only gyrA mutations. At the same time, a silent mutation (CAC to CAT) in codon 132 of gyrA gene and a silent mutation(GCT to GCG) in codon 115 of parC gene occured, which did not lead to amino acid change. Conclusion The mutations of 83 and 87 codons of gyrA and the mutatations of 87 codon of parC gene were related to fluroquinolone resistance, and the mutations of the 83 codon of gyrA gene were more important.

Objective:To investigate the antitumor efficiency of the special cytotoxic T lymphocytes(CTLs) activated by dendritic cells(DCs) pulsed with K-ras (12-Val) antigen.Methods:DCs was generated from PBMC in the presence of granuloceyte/macrophage-colony stimulating factor(GM-CSF),interleukin-4(IL-4)in vitro.DCs were differently sensitized with K-ras mutant pancreatic cancer cell line,K-ras(12-Val) mutant peptide,K-ras(12-Val) mutant peptide with the surface of cationic nanoparticle.Cell surface markers on DCs was measured by flow cytometry.The activation of CTL induced by DCs was detected by ~3H- thymidine incorporation test.The killing effects of CTL to pancreatic cancer was detected by ~(125)I-UdR release test. Production of IL-12 and IFN-γ by DCs and PBMC was detected by ELISA.Results:Compared with DCs pulsed with K-ras(12-Val) mutant peptide and K-ras (12-Val) mutant peptide with the surface of cationic nanoparticle,DCs pulsed with whole tumor antigen could better induce CTLs killing activity(P＜0.05).The DCs with K-ras(12-Val) mutant peptide and K-ras mutant peptide with the surface of cationic nanoparticle could produce specific CTL killing activity aganist pancreatic cancer cell line Patu8988(K-ras+)(P＜0.05),but not SW1990(K-ras-)(P＞0.05). K-ras (12-Val) mutant peptide with the surface of cationic nanoparticle at lower concentrations can be effectively presenting on the surface of DCs than only K-ras (12-Val) mutant peptide.Conclusion:K-ras (12-Val) mutant peptide with cationic carrier can be effectively presenting and expression of DCs and induce CTL specific killing activity aganist pancreatic cancer cell lines with K-ras (12-Val) mutant peptide.

Objective To construct prokaryotic expression plasmid of human recombinant soluble TNF-related apoptosis inducing ligand(rsTRAIL),then to investigate the effects of rsTRAIL on apoptosis in non-small cell lung carcinoma(NSCLC).Methods The encoding sequence for rsTRAIL was amplified with RT-PCR and cloned into PQE30 vector to establish the prokaryotic expression system.The competent cells of host strain of M15 were transformed by the recombinant plasmid.The expression of the target protein was induced with IPTG and purified by Ni2+-NTA agarose column.rsTRAIL was added in the media of A549 and H460wt cells,then the viability was examined by MTT assay.Apoptosis of H460wt cells was observed under fluorescence microscopy.The apoptotic rate of tumor cells was examined by FACS.Results The cloned fragment of rsTRAIL was 100% consistent with that reported in GenBank.The expressed protein with molecular weight of 21 000 in SDS-PAGE as expected was obtained and recognized by a commercial McAb.The apoptotic rate of H460wt cells after treated with rsTRAIL for 24 h was 43.2%.Cislatin enhanced the effect of rsTRAIL on A549 cells.Conclusion The rsTRAIL is obtained after Ni affinity chromatograph.rsTRAIL has a strong cytotoxic activity against NSCLC and cisplatin may enhance the antitumor effect of rsTRAIL.

Objective To construct a recombinant expression vector of human interleukin-24(hIL-24) gene and express it in E.coli M15,and to evaluate the bioactivity of IL-24 fusion protein.Methods The human IL-24 cDNA fragment was amplified from plasmid by polymerase chain reaction(PCR),and sequenced.PQE/hIL-24 was constructed by gene rearrangement,then it was transformed into E.coli M15.The expression of the target protein was induced with IPTG and purified by Ni2+-NTA agarose column.The expressed recombinant hIL-24(rhIL-24) was identified by SDS-PAGE and Western blotting.Normal peripheral blood mononuclear cells(PBMCs) were cultivated with the expression protein for 48 and 72 h,the levels of IL-6,IFN-? and TNF-? of PBMCs stimulated with rhIL-24 were detected by ELISA.Results The recombinant prokaryotic expression vector PQE/IL-24 was constructed successfully and expressed stably in E.coli M15.At about 18 400 of molecular weight,there was an induced protein band.The levels of IFN-?,IL-6 and TNF-? in the group of cultivated with the expression protein were obviously higher than those in the groups without the expresson protein(P

Objective:To investigate the expression of cyclooxygenase-2 in primary hepatocellular carcinoma, in cancer surrounding tissues and normal liver tissue and its relationship with clinical pathological features.Methods:The expression of COX-2 was detected in 30 cases of hepatocellular carcinoma, 20 cases of cancer surrounding tissue and 10 normal liver tissue by flow cytometry (FCM) and immunohistochemistry (SP). The clinical data were analyzed retrospectively.Results:(1)The expression of COX-2 in the HCC tissue was significantly higher than in cancer surrounding tissues and normal liver tissue (P0.05).Conclusion:The hyperexpression of COX-2 in tissue can reflex the biological behavior of HCC,and have very important role in the development of HCC.The specificness of COX-2 protein expression make it to be new target of tumor diagnosis and treatment.These results provide a theoretical basis for the chemoprevention of hepatoma.

Objective:To study the expression of COX-2 and Survivin in HCC tissues and their relationship for supplying experimental evidence for gene diagnosis and treatment of HCC.Methods:The expression of COX-2 and Survivin was detected in 30 cases of hepatocellular carcinoma and 10 normal liver tissue by Flow cytometry (FCM) and immunohistochemistry (SP).Results:Two experimental methods showed that the positive expression of COX-2 and Survivin in HCC was significanly higher than that in normal liver tissue and there was significanly positive correlation between the expression of COX-2 and Survivin.Conclusion:The hyper-expression of COX-2 and Survivin in tissue can reflex the biological behavior of HCC and there are synergistic effect between them during the development of HCC.A possible mechanism is inhibition of tumor cell apoptosis through upregulating Survivin by COX-2,and promoting abnormal cell proliferation in development of hepatocellular carcinoma.

Objective To study the inhibitory effects of dehydroepiandrosterone (DHEA) and its metabolites-dehydroepiandrosterone sulfate (DHEAs) on the proliferation of HepG2 and HT-29 and their mechanism.Methods HepG2 and HT-29 were incubated by DHEA or DHEAs with different concentrations (1,10,50,100 and 200 ?mol?L-1) for 8,24,48,72 h and routine culture was used as control.The inhibitory rate was detected by using MTT chromometry and BrdU assay respectively.The activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase(HMGR),glucose -6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) were examined simultaneously.Results ①MTT chromometry:DHEA with different concentrations obviously inhibited the growth of HepG2 and HT-29 cells compared with control group(P0.05).②BrdU assay:the growth of cells were significantly inhibited by DHEA with concentrations of 50,100 and 200 ?mol?L-1,especially to HepG2 cells(P0.05).Conclusion DHEA has strong anti-proliferative effects on both HepG2 and HT-29 cell lines and inhibitory effects on the activities of G6PD or HMGR,however,DHEAs has no obvious effect.

Objective To investigate the suppression to 2-glycoprotein-1-specific B cell response by human recombinant 2-glycoprotein-1 domain Ⅰ dimer(rh?2-GP1-DⅠ2) in rh?2-GP1 immunized mice.Methods The effects of treatment using rh?2-GP1-DⅠ2 on titer and ratio of anti-?2-GP1 were analyzed by solid phase ELISA.The number of splenic ?2-GP1-specific antibody-forming cells(AFC) was counted by ELISPOT.Results The levels of anti-?2-GP1 from rh?2-GP1 immunized mice treated with rh?2-GP1-DⅠ2 were significantly decreased compared with those in negative controls(P

Objective To investigate the effects of topiramate on neurons apoptosis and the hydrous capacity in brain tissue in rats with perinatal acute ischemic brain damage.Methods The perinatal acute ischemic brain damage model was established by ligation of both uterine arteries of full-term pregnant Wistar rats(n=315).They were divided randomly into topiramate one and five times groups and hypoxic-ischemia(HI) group(n=105),and normal collate group consisted of 105 normal Wistar rats.The effects of topiramate were observed by the changes of hippocampal apoptosis cells labeled in situ end TUNEL methods at different time spots(postnatal 3 h,6 h,24 h,(3 d,7 d),14 d,21 d,28 d) and the hydrous capacity in brain tissue within 7 d after birth.Results The hydrous capacity in brain tissue in administering drug one time group was remarkably less than that in HI group at 12 and(72 h after) birth,and it in administering drug five time group was remarkably less than those in HI group and administering drug one time group at 48 and 72 h after birth(P