The influenza A virus matrix protein2 gene(M2)which deleted transmembrane region was amplified by overlap extending PCR,and the multiepitope gene of hemagglutinin(HA)was PCR amplified with seven continuous synthesized segments by designing primer.The two gene segments were separately cloned into pMD18T vector to sequence analysis and prokarytic expression vector pET28a+ to construct the recombinant plasmid pET28a+M2dHA.The recombinant plasmid was transformed into E.coli BL21(DE3),and the high expression strain was obtained by screening monoclones.The recombinant protein existed as inclusion bodies,which accounted about 45% of the total cellular protein.The inclusion bodies were washed with 1% Triton X100 solution twice,and dissolved in 8 mol/L urea solution.The solution protein was purified by Ni+2 affinity chromatography,and refolded by dilution renaturation,then purified by Q Sepharose FF cation exchange column.The purity of the protein was over 90% by HPLC analysis.The result of Western blot showed it has good antigenicity and specificity.These results strongly supported for the further study of the broadspectrum influenza virus subunit vaccine.

Objective: To observe the role of rmh-TNF in enhancing the anti-angiogenesis effect of cisplatin on Lewis lung carcinoma in the mice.Methods: Lewis lung carcinoma model was established in C57BL/6 mice.Sixty model mice were randomly divided into 4 groups: control group,rmh-TNF group(1500000 U/kg),cisplatin group(6.15 mg/kg), and rmh-TNF plus cisplatin group.Twelve days after implantation of cancer cells,different drugs were injected intra- tumorallv for 3d.The expression of hypoxia inducible factor-1?(HIF-1?)gene in the tumor was identified by RT-PCR. Immunohistochemistry(IHC)image analysis was performed to determine the vascular endothelial growth factor(VEGF) and kinase domain region receptor(KDR)expression and the microvessel density(MVD).Expression of matrix metallo- proteinase-2(MMP-2)was detected by flow cytometry.Results: The MVD values in the control group,the rmh-TNF group,the DDP group and the combination group were(24.76?1.28),(18.95?1.22),(19.53?1.15),(10.43?1.05),respectively,with those of the rmh-TNF and DDP groups significantly lower than that of the control group and higher than that of the combination group(all P

Eukaryotic expression plasmid pCI-neo-mdr1 which contains human multidrug resistance gene 1(mdr1),was constructed and transferred into human hepatocarcinoma HepG2 cells by use of liposome. G418 was used to screen the cells successfully with mdr1 and the selected cells was named HepG2/mdr1 Morphological and biological properties of HepG2/mdr1 cells were observed. The results show that the constructed HepG2/mdr1 cell line was high efficient and stationary in the expression of mdr1. The work was valuable and desirable for the establishment of multidrug resistant cell models,and for the study of MDR in human hepatoma. Furthermore,the work also provided a perfect model for the research of relationship between insulin resistance and MDR in hepatocarcinoma cells.

Four kinds of ionic liquids were adopted to analyze the content of rubimaillin and alizarin in Rubia cordifolia roots with ultrasonic-assisted extraction coupled with HPLC. The chromatographic column, Purospher star RP-C18 (4.6 mm x 250 mm, 5 microm), was used. Methanol and 0.4% acetic acid-water as mobile phase with flow rate at 0.85 mL min(-1), gradient elution, detection wavelength at 250 nm, chromatographic column temperature was controlled at room temperature. The result showed that rubimaillin and alizarin had the highest extraction yield when the [ HMIM] PF6methanol solution concentration of 0.6 mol x L(-1) as extraction solvent and the conditions were solid-liquid ratio of 1:80 (g x mL(-1)). Under the optimal extraction conditions, the content of alizarin from 0.01 to 0.04 microg showed a good linearity (r = 0.9999), the average recovery was 97.12%, the content of rubimaillin from 0.41 to 1.35 microg showed a good linearity (r = 0.9999), the average recovery was 98.10%. This experiment adopted environmentally friendly reagent as extraction solvent, the extraction efficiency was improved, and the environmental pollution caused by organic solvent was avoided, the harm of human body aslo was reduced. This method was simple and reliable, its repeatability was also very good, which had an important significance in the study of traditional Chinese medicine active ingredient extraction methods.
Anthraquinones ; analysis ; Chromatography, Reverse-Phase ; methods ; Ionic Liquids ; chemistry ; Pyrans ; analysis ; Rubia ; chemistry ; Ultrasonics

Objective To survey the treatment stares and clinical features of hospitalized patients with acute myocardial infarction (AMI) of 13 hospitals in Beijing in 2005.Methods Uniform questionnaires were used to register AMI patients hospitalized from January 1 to December 31,2005 in the 13 hospitals including traditional Chinese medicine (TCM) hospitals (n = 6) and western medicine hospitals (WM,n=7) from Beijing.A total of 1663 AMI patients were registered (1366 cases in WM hospitals and 297 cases in TCM hospitals).An Access database was established and patient informations were inputed,the clinical features and treatment status of hospitalized AMI patients were analyzed.Results The mean age was (63.9±12.8) years old[(62.8±12.8) years for WM Hospitals and(69.1±11.8) years for TCM hospitals,P＜0.05],male to female ratio was 2.4 :1 (2.7:1 for WM hospitals and 1.6:1 for TCM hospitals,P＜0.05).The median time to hospital was 14 hours in TCM hospitals and 11 hours in WM hospitals (P＞0.05).Incidences of history of cerebrovascular disease,high blood pressure,diabetes,hyperlipidemia and complications such as in-patient arrhythmia,cardiac insufficiency,cardiogenic shock were significantly higher in TCM hospitals than in WM hospitals.The total mortality of 1663 AMI cases was 8.2% (15.8% in TCM hospitals vs.6.6% in WM hospitals,P＜0.01).The reperfusion rate including emergency PCI and thrombolytic therapy rate was 31.3% in 13 hospitals (33.3% in WM hospitals vs.21.9% in TCM hospitals,P ＜0.05).Percent of guideline recommend drug use for AMI was as follows:aspirin 93.6%,ACEI and ARB 85.1%,β-blocker 78.7%,low molecular weight heparin 85.4%,statins 74.7%.Conclusions Reperfusion therapy and guildline recommended drugs were widely used although there was a need for further improvement.The hospitalized mortality showesd a downward trend compared with results from five years ago,patients in TCM hospitals had an independent clinical features.

No abstract available.
Anticoagulants ; Echocardiography, Transesophageal ; Embolism ; Endocarditis ; Endocarditis, Bacterial ; Heart Valve Prosthesis ; Heart Ventricles ; Hemodynamics ; Male ; Mitral Valve ; Mitral Valve Insufficiency ; Outpatients ; Pneumonia ; Systolic Murmurs ; Warfarin ; Humans

Objective To study the efficacy and security of postoperative radiotherapy alone and post-operative radiotherapy combined with chemotherapy in patients with high-grade cerebral glioma.Methods Fifty-nine patients with high-grade cerebral glioma confirmed by pathology, including 35 with grade Ⅲ and 24 with grade Ⅳ, admitted to our hospital from June 2004 to June 2008, were chosen in our study; these patients were randomly divided into 3 groups (A, B and C). All the patients underwent three-dimensional conformal radiation therapy (3D-CRT) with a dose of 54-66Gy/27-33f/5-7w by 6/10MV-X ray; and the median total dose was 60 Gy; group A (n=21, including 13 with grade Ⅲ and 8with grade Ⅳ) received chemotherapy with termozolomide (TMZ) at a dosage of 75 mg/m2·d during radiotherapy, following by 150-200 mg/ (m2·d) for 5 d, and enjoying 28 d per cycle for a total of 3-5cycles; group B (n=26, including 14 with grade Ⅲ and 12 with grade Ⅳ) received chemotherapy with the regimen of nimustine (ACNU) with a dosage of 90 mg/m2 on the 1st d and teniposide (VM-26) at a dosage of 60 mg/m2 on the 1st, 2nd and 3rd d, enjoying 6-8 weeks per cycle for a total of 4-6 cycles; group C (n=12,including 8 with grade Ⅲ and 4 with grade Ⅳ) received radiotherapy alone. Clinical evaluations of tumor response and adverse effects were performed periodically. The primary end points were disease progression-flee survival (PFS) and overall survival (OS) Results The effective rate in the group A, B and C was 81.0％, 71.4％ and 33.3％, respectively, indicating that significant differences exited between group C and both group A and B (P＜0.05). The incidence of gastrointestinal side effects and toxic response in the group B was obviously higher than that in the group A (P＜0.05). The 1-, 3- and 5-year survival rates in group A were 66.7％, 19.0％ and 9.5％, respectively, which were the highest among the 3groups; those in group B were 53.8％, 15.4％ and 3.8％, respectively; those in group C were 25％, 8％ and 0％, respectively. The PFS in all the patients was 8 months and OS was 15 months; log-rank test indicated that significant differences of PFS and OS existed between patients received concomitant chemoradiotherapy and radiotherapy (X2=10.710, P=0.005; X2=7.185, P=0.028); the incidence of PFS and OS in group A and B was significantly higher than that in group C (P＜0.05). Conclusion To post-operative patients with high-grade cerebral glioma, concomitant chemoradiotherapy can improve the effective rate and extend the PFS and OS. TMZ is recommended as the concomitant chemotherapy regimen, having similar therapy effect with ACNU plus VM-26, but enjoying less adverse effects.

OBJECTIVETo explore the relationship between abdominal fat volume and obstructive sleep apnea hypopnea syndrome in obesity people.

METHODSFrom July 2009 to July 2010, 50 patients with BMI > 25 were prospectively selected for study from the patients who complained of snoring in the Respiratory department. The patients were divided into OSAHS group and non-OSAHS group according to the result of sleep apnea monitoring. All the patients also received full abdominal CT and the whole abdominal fat volume was measured by 3-D CT reconstruction system. SPSS 13.0 was used for statistical analysis.

RESULTSThe whole abdominal fat volume in the two groups was analyzed by T- test, which was significantly different between the two groups (P < 0.01). It showed that there was a statistical relationship between OSAHS and abdominal fat in obesity people.

CONCLUSIONIn obesity people, OSAHS has a close relationship with abdominal fat volume. The abdominal fat volume is markedly higher in OSAHS patients than that in non-OSAHS people.

Abdominal Fat ; diagnostic imaging ; Adult ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Obesity ; diagnostic imaging ; Prospective Studies ; Radiography ; Sleep Apnea, Obstructive ; epidemiology

OBJECTIVETo explore a technology for diagnosing VHL mutations from a single cell and provide experimental evidences for the feasibility of applying technology in detecting genetic mutations from a single cell.

METHODSAfter whole genome amplification (WGA) based on multiple displacement amplication (MDA) for a single cell, we did regular PCR following sequencing and detected the genotypes using the real time PCR based on TaqMan probes. We detected VHL mutations by the different terminal fluorescent changing.

RESULTSThe rate of amplification for single cell based on MDA was 90.91%. The rate of contamination was 0. After sequencing, the allele drop out (ADO) rate of heterozygotes was 26.67%(8/30); combined with the different terminal fluorescent changing, the rate of ADO of heterozygotes was 16.67%.

CONCLUSIONWGA based on MDA for a single cell followed by regular PCR with sequencing and real time PCR can specially and accurately detect the VHL genotypes of single cells.

Base Sequence ; DNA Mutational Analysis ; Female ; Genotype ; Humans ; Lymphocytes ; metabolism ; pathology ; Middle Aged ; Molecular Sequence Data ; Mutation ; Polymerase Chain Reaction ; Preimplantation Diagnosis ; Von Hippel-Lindau Tumor Suppressor Protein ; genetics ; von Hippel-Lindau Disease ; blood ; genetics

OBJECTIVETo investigate the effect of autocontrol micromotion locking nail (AMLN) on experimental fracture healing and its mechanism.

METHODS16 goats undergoing both sides of transverse osteotomy of the femoral shafts were fixed intramedullary with AMLN and Gross-Kempf (GK) nail, respectively. The follow-up time was 7, 14, 28 and 56 days. Roentgenographic, biomechanical, histological, scanning electromicroscopic and biochemical analyses were done.

RESULTS(1) The strength of anticompression, antiflexion and antitorsion in the fractural end in the AMLN-fixed group was higher than that of GK nail-fixed group; whereas, the rate of stress shelter in the fractured end decreased significantly (P<0.01). (2) The content of the total collagen, insoluble collagen, calcium and phosphate in the AMLN-fixed group was higher than that in the GK nail-fixed group (P<0.05). (3) Histological observation and quantitative analysis of calluses revealed that AMLN could promote the growth of bridge calluses and periosteum calluses. Hence the fracture healing and remolding process achieved early, which was much better than traditional GK nail fixation. (P<0.05). (4) 7-14 days postoperation, the calluses of AMLN-fixed group was flourish and camellarly arranged and the collagen fibril formed constantly in the absorption lacuna of bone trabecula. 28-56 days postoperation, the collagen fibril was flourish around the absorption lacuna and was parallel to the bone's longitudinal axis. Active bony absorption and formation were seen, so was remolding and rebuilding. Haversian system was intact and the bony structural net was very tenacious because of the deposition of calcium salt. None of the above findings was observed in the GK nail-fixed group.

CONCLUSIONSThe design of AMLN accords well with the plastic fixation theory. As the geometry ametabolic system constituted by the intramedullary fixation instruments and the proximal and distal end of the fracture is very firm and stable, the disturbance to the physical stress distributed in the fractural end is light. The generation and conduct of the intermittent physical stress between the fractural parts could reach the balance between stress conduct and stress protection. The feature that the healing and remolding take place at the same time speeds up the fractural healing process.

Animals ; Biomechanical Phenomena ; Bone Nails ; Femoral Fractures ; surgery ; Fracture Fixation, Intramedullary ; instrumentation ; Fracture Healing ; physiology ; Goats ; Microscopy, Electron, Scanning ; Stress, Mechanical