Objective To study the effect of lgG Fc gene on JEV DNA vaccine immunity. Methods Gene encoding IgG Fc was amplified by nested-RT-PCR technique from BALB/c murine spleen cells. JEV prME protein gene was obtained with restriction endonuclease BamH Ⅰ/EcoR Ⅰ from the eukaryotic recombinant named after pJME, which was constructed by us before. Recombinant, named after pJME/IgG Fc, with above two genes encoding JEV prME protein and BALB/c murine IgG Fc was constructed, and was tested by restriction enzymes analysis and DNA sequencing, then was transfected into China hamster ovary (CHO) cells by Lipo-fectAMINE 2000. Distribution and expression of the fusion proteins encoded by JEV prME protein and BALB/c murine IgG Fc genes in transfected CHO cells were detected by immunofluorescence and Western blot. The BALB/c micc were vaccinated with pJME/IgG Fc via intramuscular injection. Then the cytotoxic T lymphocyt (CTL) activity were assessed by lactic dehydrogenase (LDH) and the neutralizing antibody titer were assessed by 80% plaque reduction neutralization test. Results Molecular weights (2001 bp, 2730 bp) of the two in- serts released from pJME/IgG Fc with two group of restriction analysis associated with BamH 1/EcoR I and BamH Ⅰ/Not Ⅰ were correlated to the expected theoretic results respectively. It was estimated that molecular weight (Mr) of the fusion protein was 101 x 103. The expression of the above fusion protein was mainly distribu- ted in endochylema of transfected CHO cells,and not much in membrane of transfected CHO cells. CHO cells transfected with pJME/IgG Fc could express the fusion protein at the 32th cell passage. After immunization, the CTL activity and the neutralizing antibody titer in the pJMF/IgG Fc vaccinated group increased significantly compared with other vaccinated groups(P <0.05). Conclusion The recombinant pJME/IgG Fc was construc- ted and transfected into CHO cells successfully, and CHO cellular lines expressed fusion protein encoded by JEV prME protein and BALB/c murine lgG Fc genes stably were obtained. IgG Fc gene could reinforce the cellular immunity and humoral immunity of JEV DNA vaccine.

This study aimed to investigate the immune adjuvant effect and mechanism induced by chitosan nanoparticles carrying pJME/GM-CSF. In this study, plasmid DNA (pJME/GM-CSF) was encapsulated in chitosan to prepare chitosan-pJME/GM-CSF nanoparticles using a complex coacervation process. Immunohistochemistry was used to detect the type of infiltrating cells at the site of intramuscular injection. The phenotype and functional changes of splenic DCs were measured by flow cytometry after different immunogens were injected intramuscularly. The killing activity of CTLs was assessed using the lactate dehydrogenase (LDH) release assay. The preparation of chitosan-pJME/GM-CSF nanoparticles matched the expected theoretical results. Our results also found that, after pJME/GM-CSF injection, the incoming cells were a mixture of macrophages, neutrophils, and immature DCs. Meanwhile, pJME/GM-CSF increased the expression of MHC class II molecules on splenic DCs, and enhanced their Ag capture and presentation functions. Cell-mediated immunity was induced by the vaccine. Furthermore, chitosan-pJME/GM-CSF nanoparticles outperformed the administration of standard pJME/GM-CSF in terms of DC recruitment, antigen processing and presentation, and vaccine enhancement. These findings reveal that chitosan could be used as delivery vector for DNA vaccine intramuscular immunizations, and enhance pJME/GM-CSF-induced cellular immune responses.
Adjuvants, Immunologic ; administration & dosage ; Animals ; Chitosan ; administration & dosage ; immunology ; Dendritic Cells ; immunology ; virology ; Encephalitis Virus, Japanese ; genetics ; immunology ; Encephalitis, Japanese ; immunology ; prevention & control ; virology ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; administration & dosage ; genetics ; immunology ; Humans ; Immunity, Cellular ; Japanese Encephalitis Vaccines ; administration & dosage ; genetics ; immunology ; Mice ; Mice, Inbred BALB C ; Nanoparticles ; administration & dosage ; Spleen ; immunology ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Vaccines, DNA ; administration & dosage ; genetics ; immunology

Aim To investigate the inhibitory effects of zacopride(Zac) on arrhythmia induced by isoproterenol ( ISO) and the underlying mechanisms in rats. Meth-ods ①ECGs were recorded in anesthetized rats in vi-vo to observe the effects of zacopride on arrhythmia in-duced by ISO. ② Intracellular microelectrode tech-nique was used to investigate the effects of zacopride on resting membrane potential, delayed afterdepolariza-tions ( DADs) and triggered activity ( TA) induced by ISO combined with 3. 6 mmol·L-1 CaCl2 in right ven-tricular papillary muscle of rats. Results ① In ISO group rats, ventricular premature beats ( VPB ) oc-curred frequently with ST-segment depression. Com-pared with ISO group, the incidence of VPB in ISO+Zac group decreased from 100% to 50% ( n=6 , P<0. 05 ) and the total number of VPB recorded in 1 hour significantly reduced from 1 574 ± 521 to 33 ± 40 ( n=6,P<0. 05). ② Zacopride at 1 μmol·L-1 could hy-perpolarize the resting membrane potential of right ven-tricular papillary muscle in normal rat from ( -74. 42 ± 1. 95 ) mV to ( -78. 50 ± 2. 07 ) mV ( n =6 , P <0. 05). ③ Zacopride at 1 μmol·L-1 significantly de-pressed the DADs and TA induced by ISO combined with 3. 6 mmol·L-1 CaCl2 in right ventricular papilla-ry muscle. The incidence of DADs decreased from 93. 75% in rats in ISO group to 25% in ISO +Zac group ( n =16 , P <0. 05 ) , and this antiarrhythmic effect could be reversed by 1 μmol·L-1 BaCl2 . Conclusions Zacopride, a selective IK1 channel ago-nist , can significantly inhibit cardiac arrthymia induced by ISO in rats, the mechanism of which is mainly at-tributed to zacopride-induced hyperpolarization of the resting membrane potential and subsequent suppression of DADs and TA via enhancing IK1 . These results pro-vide further evidence that to enhance IK1 moderately may be a feasible pathway for antiarrthymic therapy.

Endoproteinase Lys-C/trypsin sequential digestion and trypsin digestion were used in 293 T cell proteomics sample preparation and the results of Lys-C/trypsin sequential digestion and trypsin digestion in proteomics sample preparation was systematically evaluated. It was found that the number of identified peptides and proteins increased significantly, and missed cleavage sites, especially K sites decreased dramatically through Lys-C/trypsin sequential digestion. And the average sequence coverage of identified proteins in Lys-C/trypsin sequential digestion sample was higher than that in trypsin digestion sample. Besides, different amount of enzymes was tested to select the optimal usage of enzymes in Lys-C/trypsin sequential digestion. This study provided the references for proteomics sample preparation.

Psychiatric professional talents are specially needed in China.Attention should be paid to the cultivation of clinical thinking ability in the process of cultivating professional master degree postgraduate majoring in mental health and psychiatry.Following measures can be taken such as using problem-based leaming(PBL) interactive teaching,organizing symposium,urging students to reviewing the language knowledge and logic knowledge,implementing specialized training to promote students' rigorous logical thinking ability,the ability to get information and interpersonal communication ability,strengthening the clinical practice teaching and paying close attention to the analysis and summary after the failure.

Objective To study the effects of acetylcholine combined with vincristine (VCR) on A549 cells, furthermore to study the influence that is due to affecting the expression of the two proteins bcl-2 and p53. Methods MTT assay was used to analyze growth inhibition effect of acetylcholine alone and combined with IC50VCR on A549 cells.The expression of bcl-2 and p53 was measured by immunohistological chemistry technique(IHC) and Western blot.Results The inhibition rates for A549 cells with 0.1,1 and 10 μmol/L acetylcholine were (0.050±0.032)％,(0.101±0.021)％,(0.169±0.015)％.The inhibition rates for tumor cells with 0.1,1 and 10 μmol/L acetylcholine joint 0.2 μg/ml VCR were (0.529±0.023)％,(0.545±0.011)％,(0.589±0.015)％,the difference was statistically significant compared with the VCR group (q'values were 1.09,1.37,1.83,P＜0.05).Acetylcholine alone exerted inhibitory effect on A549 cells in a concentration dependent manner and significanrtly enhanced its sensitivity to VCR (P＜0.05).acetylcholibne (0.1-10 μmol/L)combined with IC50VCR decrased the expression of bcl-2 and p53 (P＜0.05).Conclusion Acetylcholine alone and combined with VCR can inhibit the growth of A549 cells. Significant synergistic effect between acetylcholine and VCR is found in inducing cell apoptosis by changing the expression of bcl-2 and p53.

Objective Construction and expression of recombinant plasmid fusing encoding prME protein derived from Japanese encephalitis virus （JEV） and GM-CSF of BALB/c mouse. Methods GM-CSF gene was ampli/ied by nested-RT-PCR from BALB/c spleen cells. JEV prME protein gene was eluted by the digestion with restrict/on endonucleases BamH I/EcoR I from pJME piasmid. Genetic fusion of prME protein and GM-CSF are subcloned info pcDNA3.1 （＋） eukaryotic vector, and named as pJME/GM-CSF.The recombinant was confirmed by restriction enzymes digestion and DNA sequencing, and then transfected into China hamster ovary （CHO） cells by Lipofectamine2000. pJME/ GM-CSF expression in CHO cells was examined by Western blot. Results We observed 2001 bp and 2472 bp DNA fragments when pJME/GM-CSF was digested with BamHI/EcoRI and BamHI/NotI respectively as expected. The estimated molecular weight of the fusion protein was 85kD. Conclusion The recombinant pJME/GM-CSF was constructed and transfected into CHO cells successfully with pJME/GMCSF stably expressed.

To study the traditional Chinese medicine (TCM) syndrome distribution in patients with hepatitis B virus (HBV) infection in Qidong region of Jiangsu Province, China.

Objective To assess the safety, feasibility, short-and mid-term efficacy of wingspan stent for treating patients with symptomatic intracranial artery stenosis. Methods A total of 113 patients with severe symptomatic intracranial stenosis were enrolled and Gateway-wingspan stenting were performed on all patients. The technical success, the pre- and post-stenting stenosis, perioperative complications, clinical outcome and restenosis rates were recorded, and chi-square test was used for analysis of complication rate by comparing our results with the results of Warfarin-Aspirin Symptomatic Intracranial Disease (WASID) study and NIH multi-center Wingspan stenting trial. Results The technical success rate was 99. 1% ( 112/113). The mean pre and post-stent stenoses were (80.7 ± 9.3)% and (27.7 ± 9.7)% (χ2 =9.397,P < 0. 05 ). The total complication rate was 4.4% (5/113 ) during the follow-up ( mean 14. 5 months, range 1-28 months), and the frequency of restenosis was 12. 5% (5/40) at 6 months. The primary endpoint events, ischemic stroke, and lesion-related ischemic stroke were lower in our study (4.5%, 3.5%,3.5% ) compared with the results of WASID trial (21.1%, 20. 4%, 15.0% ,P<0. 05). For those with poor outcome in the three high-risk sub-groups which were with more than 70% stenosis, or last event from the treatment was less than 17 days, or NIHSS was above 1, a better outcome was observed in our group (4. 5% ,4. 7% and 2. 0% in our study, 19.0%, 17.0% and 19. 6% in previous study, P < 0. 05). The medium-term efficacy in this group (4. 5% ) significantly improved compared with NIH study ( 14. 0% ,P <0. 05 ). Conclusions Wingspan stenting for symptomatic intracranial arterial stenosis is with good safety,feasibility and low perioperative stroke rate and mortality. The incidence of primary endpoint events and the ischemic events are lower than those of medication group, and the efficacy of stenting is significantly better than medication even in high-risk population.

ObjectiveTo compare two treatment methods for acute iliofemoral vein thrombosis:c atheter-directed pharmacomechanical thrombolysis (CDPT,47 cases) and intervention combined surgicaltherapy( IST,14 cases).MethodsThis study includes 61 patients of acute iliofemoral vein thrombosis treated by CDPT or IST.All discharged cases were followed up by telephone for a period of 14 -37 months.ResultsAmong the 61 patients (64 extremities),47 (forty-seven extremities) treated by CDPT,and 14 cases (seventeen extremities) treated by IST.The IST group included three patients of bilateral iliofemoral vein thrombosis,five patients on postoperative status within one month,and three patients in which the iliofemoral vein was not accessible.When discharged from hospital,the effective rate of edema relief is 93.6％ in CDPT group while that is 94.1％ in IST group; Melena occurred in one patient of CDPT group and incision hematoma occurred in one patient of IST group.According to the results of 14 -37 months follow-up,the effective rate of edema relief is 85.0％ in CDPT group while that is 85.7％ in IST group ( x2 =0.004 and the P =0.948).Calf pigmentation occurred in only one patient of CDPT group.The patency rate of vein by BUS examination is 52.6％ in CDPT group while that is 84.6％ in IST group x2 =4.157,P =0.041 ).ConclusionsComparing with CDPT group,IST group has the similar effective rate of edema relief,but has higher patency rate of iliofemoral vein.In case of bilateral acute iliofemoral vein thrombosis,in patients in whom thrombolysis is contraindicated,or when the iliofemoral vein is not accessible,IST is the treatment of choice for acute iliofemoral vein thrombosis.