Aim To evaluate the effect of expression inhibition of spermine oxidase(SMO)on the actitumor activity of polyamine analogue CPENSpm (N~1-cyclopropylmethyl-N~(11)-ethylnorspermine).Methods siRNA technique was used to inhibit expression of SMO in human lung cancer line A549.QT-RT-PCR and enzyme activity assay was performed to determine the expression level of SMO.The cell proliferation was detected by MTT assay.The apoptosis of A549 cells were evaluated by DNA degradation and Sub-G_1/flow cytometry assay.Results The A549 cell line with silenced SMO expression was successfully obtained.Basic SMO mRNA and enzyme activity levels in the SMO-siRNA plasmid transfected cells were 0.53% and 14% lower than that in the control cells respectively. Treating A549 control cells by 10 μmol·L~(-1) CPENSpm for 24 hours resulted in a 10-folds up-regulation of SMO in mRNA level and 20-fold increase in enzyme activity,but this drug-induced SMO expression was obviously prevented in SMO-siRNA plasmid transfected cells.MTT assay demonstrated that SMO expression inhibition decreased the sensitivity of A549 cells to CPENSpm exposure(0～20 μmol·L~(-1)).DNA degradation and sub-G_1 assay proved a deceased ability of CPENSpm to induce apoptosis in SMO-siRNA plasmid transfected cells.Conclusion Up-regulation of SMO by CPENSpm is possibly one of the molecular basics for its antitumor activity.

Objective To construct markless gene deletion mutant at the clpP loci on the chromosome of Streptococcus mutans(S.mutans).Methods ASp resistance gene was amplified by PCR,to construct the Sp resistance cassette where the Sp resistance gene was flanked with two loxP site.After the clpP gene was cloned into the pGEM-T-Easy TA cloning vector,it was digested and linked with the Sp resistance cassette,yielding homologous recombination vector pIB △ clpP-Sp.The vector was linearized and used for the transformation of S.mutans UA159,with transformants selected on TPY plates containing Sp.The selected strain was transformed with the thermosensitive plasmid pCrePA to excise the Sp resistance gene.The pCre-PA was then easily eliminated at nonpermissive temperature,resulting in a markless mutant strain carrying a deletion at the clpP loci,which was verified by PCR and DNA sequencing.Results The result of the PCR analysis and DNA sequencing indicated that a part of the clpP gene was deleted.There was a loxP at this loci without the Sp resistance gene.Conclusion The markless clpP-deletion mutant of S.mutans was constructed successfully,which laid a foundation for further study of its biological function and its influence on the cariogenicity of S.mutans.

Objective To investigate whether difluromethylornithine (DFMO) can be used in the treatment of human leukemia. Methods The cell proliferation was detected by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)] assay after treatment of human lymphocyte Jurkat cells by DFMO (0 to 10 mmol/L) for 24 to 72 h. Enzyme activity of spermine oxidase (SMO) and acetylpolyamine oxidase (PAO) was determined by chemiluminesence assay. DNA fragmentation assay was used to evaluate cell apoptosis. Fluorescent dye assay was performed to determine the changes in mitochondrial membrane potential. Western blotting was used to determine Bax content. Casepase-3 enzyme activity was measured by spectrophotometric method. Results DFMO treatment inhibited the proliferation of Jurkat cells significantly in a dosage- and time-dependent manner (P

AIM: To prepare recombinant human spermine oxidase (SMO) and polyclonal antibody against human SMO by gene recombination techniques. METHODS: Human SMO cDNA was amplified from total RNA of A549 cells through reverse transcription PCR. The cDNA was then cloned into pET-15b to construct SMO prokaryotic expression vector. After transforming, the vector was induced to express recombinant SMO by IPTG in E. coli BL21 (DE_3). Recombinant SMO was purified by Ni-NTA resin under denaturing condition and then was dialyzed to renature. The enzyme activity of recombinant SMO was analyzed by chemical fluorescent method. SMO polyclonal antibody was prepared by using recombinant human SMO protein purified by polyacrylamide gel electrophoresis as antigen to inoculate rabbit intradermally. The titer and specificity of anti-sera were determined by ELISA, Western blot and Immune Cell Chemistry. RESULTS: Purified and dialyzed recombinant human SMO has the specificicity of oxidizing the spermine. The polyclonal antibody has high titer and specificity against human SMO. CONCLUSION: This research established a method for prokaryotic expression, purification and polyclonal antibody preparation of human SMO. The method lays a foundation for the future functional research of SMO.

Objective: To evaluate the anti-tumor and side effects of activated-HLA haploidentical peripheral blood tem cells (haplo PBSCs) in the treatment of advanced refractory solid tumor patients. Methods: Forty-two patients with advanced refractory tumor, who were diagnosed in our hospital from Oct. 2004 to Oct. 2007, were enrolled in this study (all patients signed informed consent), including 12 with ovarian cancer, 9 with renal cancer, 8 with lung cancer, 8 with breast cancer, 2 with colon cancer, 2 with gastric cancer, and 1 with melanoma. The donors were healthy direct relatives of the patients; the donors' haplo-PBSCs were mobilized, collected, and activated by rhIL-2 in vitro. The clinical efficacy and side effects of haplo-PBSCs therapy were assessed by CT/PET-CT scanning, RESIST standard, KPS score, and clinical response rates, etc. Results: All 42 patients received one episode of haplo-PBSCs treatment. The progression-free survivals (PFS) were 6 months and the clinical beneficial rate (CR+PR+SD) was 73.8%. The beneficial rate of life quality was 76.2% and the KPS increased by 20 (0-30) points on average after haplo-PBSCs treatment. The patients with KIR unmatched in GVH direction had better outcomes than those with KIR matched or KIR unmatched in HVG direction (P<0.05), and the clinical beneficial rate, PFS and total beneficial rate were 94.1% vs 60.0%, (13.4±1.3) vs (8.0±0.9) months, and 89.5% vs 65.2%, respectively (all P<0.05). The donor/recipient relation as the mother/child had a better outcome than that as the father/child (P<0.05). Patients with renal cancer or ovarian cancer had better outcomes than those with other cancers, with clinical beneficial rates being 90.0% and 81.8%, respectively. Conclusion: Activated haplo-PBSCs therapy can induce non-specific anti-tumor effect, and improve the clinical symptom and life quality of advanced tumor patients.

Objective　To evaluate therapeutic effect and opportunity of laser in situ keratomileusis(LASIK) for treatment of hypermetropia.Methods　Three hundred and ninety hypermetropia eyes were treated with LASIK using solid excimer laser instrument(made in Novatec Corporation, USA),and were reexamined at 1,3,6 and 12 months after LASIK.Results　Therapeutic effect of LASIK was more remarkable, stable and reliable, that of other methods in treating +2.00～+10.00D hypermetropia and astigmatism, and the rate and degree of refraction regression were small. The diopter has been stable in 6～12　months after LASIK. Diopter of 87 percentage of all eyes≤+2.00D, and 61 percentage≤+1.00D. LASIK had shorter recovery time, more gently operation reaction, more remarkable droping of diopter than other methods. Elevation of vision was remarkable after LASIK. Elevation of vision of amblyopia children who were treated with LASIK was quicker and more remarkable after treatment of amblyopia than that of other amblyopia children who were not treated with LASIK.Conclusion　At present, LASIK is the best therapeutic method to treat hypermetropia.

Phospholipase D(PLD) is ubiquitous in bacteria,fungi,and mammal.In pathogenic microorganisms,PLD can be pathogenic determinant and play a role in spore generation.In mammalian cells,PLD functions in several signal transduction pathways,such as membrane transportation,mitosis regulation,and actin cytoskeleton regulation.In the process of pathogens invasion host cells,both of the pathogen and host cells’ PLD will be activated and a series of cascade reaction will be generated.During this process,pathogen’s PLD can regulate the polymerization and reorganization of its own actin filaments and induce the polymerization or reorganization of the host cell actin filaments near the foci,thus to promote the phagocytosis of the pathogen by host cell.Investigating the role of PLD activation in the infection will be significance for further understanding the molecular mechanism of pathogen-host cell interaction.

8 ?g/ml were 7 strains in prophylactic treatment group and 3 strains in non-fluconazole prophylactic treatment group respectively.The two groups had significant difference (x~2=8.75,P

Objective: To investigate the effect of RetroNectin on CIKs cells and the related mechanisms. Methods: Peripheral blood mononuclear cells (PBMC) were collected from patients and divided into two groups: group Ⅰ and group Ⅱ. Samples in group Ⅰ were seeded into culture flask precoated with RetreNec-tin and CD3mAb to induce CIKs. While samples in group Ⅱ were seeded into common culture flask. The pro-liferation of CIKs was detected by cytometric analysis. The cytotoxic activity of CIKs was determined by LDH assays. The phenotype changes and cell cycle of CIKs were identified by flow cytometry. The apoptosis of cells was detected by Annexin V/PI. Western blot was employed to detect the level of protein Vav1. The CD49d and CD49e were blocked by anti-CD49d and anti-CD49e and the proliferation of cells was tested by cytometric analysis after the blockage. The phenotype changes of cells were identified by flow cytometry after the blockage. Results: RetroNectin enhanced the proliferation of CIKs (P<0.05). Flow cytometric analysis showed that RetroNectin significantly increased the number of CD25+ T cells (P<0.05). RN-CIK was more ac-tive than CIK in killing HCT-8 cell lines in vitro (P<0.05). RetroNectin could block the CIKs at G_1 phase (P<0.05) and resist apoptosis. There was no significant difference in the proliferation between the two groups af-ter the blockage with CD49d and CD49e (P>0.05). The expression of protein Vavl was associated with CD25+T cells. Conclusion: RetroNectin enhances the proliferation of CIKs by influencing the cell cycle, resist-ing apoptosis possibly through the site of CD49d and CD49e, and inducing T cell activation as the second sig-naling through Vav1.

Objective To investigate the differences in tumor volume and metastatic tumors of the liver and regional lymph nodes between contrast-enhanced computed tomography (CT) and diffusion-weighted magnetic resonance imaging (DWMRI) through a comparative analysis,as well the useful information for target volume delineation,and to guide radiotherapy in clinical practice.Methods A total of 40 patients with pancreatic cancer were enrolled and underwent contrast-enhanced CT and DWMRI in the same position.The target volume was delineated,the major axis of the maximum tumor section was measured,and the numbers of liver metastatic tumors and metastatic tumors of the lymph nodes with a diameter of 5-8 mm or＞8 mm were measured based on the CT and DWMRI images.The analysis was performed by using paired t-test or paired Wilcoxon rank sum test.Results The mean gross tumor volume (GTV) delineated by contrast-enhanced CT and DWMRI was 54.95 cm3 and 41.67 cm3(P =0.000),and the mean value-different value was 13.28 cm3.The major axis of the maximum tumor section measured by contrast-enhanced CT and DWMRI were 4.18 cm and 3.94 cm (P=0.000),respectively,and in two patients,dCT was smaller than dDWMRI.A total of 83 liver metastatic tumors were identified by contrast-enhanced CT,and 112 were identified by DWMRI;the liver metastatic tumors detected by contrast-enhanced CT accounted for 74％ of those detected by DWMRI.As for the metastatic tumors of the lymph nodes with a diameter of 5-8 mm or＞8 mm,103 or 46 were detected by contrast-enhanced CT,and 200 or 56 were detected by DWMRI,and the tumors detected contrast-enhanced CT accounted for 52％ or 82％ of those detected by DWMRI.There were significant differences in all data between contrast-enhanced CT and DWMRI.Conclusions GTV and the major axis of the maximum tumor section measured by DWMRI are lower than those measured by contrast-enhanced CT,and contrast-enhanced CT is sensitive in detecting the metastatic tumors of the liver and lymph nodes.However,it is necessary to conduct further controlled experiments with reference to pathology.