OBJECTIVE To investigate the influence of short hairpin RNA targeting human telomerase reverse transcriptase(hTERT)on inhibition of telomerase activity and on expression of protein c-myc in nasopharyngeal carcinoma cells. METHODS Plasmid shRNA1 containing fluorescein gene and hTERT cDNA sequences were synthesized. Cells were transfected with plasmid shRNA1. The cell viability was examined using the MTT assay. The activity of telomerase was tested by polymerase chain reaction telomeric repeat amplification protocol- enzyme-linked immunosorbent assay (PCR-TRAP- ELISA),protein c-myc expression was tested by western blot. RESULTS It was observed that treatment with pshRNA1 in the presence of a valid transfection reagent could significantly reduce telomerase activity and the expression of protein of c-myc. CONCLUSION Inhibition of telomerase activity or expression of hTERT mRNA in nasopharyngeal carcinoma cells could inhibit cells proliferation and reduce the expression of protein of c-myc.

BACKGROUND:Tetramethylpyrazine (TMP) can inhibit the expression of vascular endothelial growth factor (VEGF), but it is uncertain that TMP inhibit the growth and proliferation of HL-60 leukemic cells induced by VEGF.OBJECTIVE:To observe the effect of TMP on the proliferation of HL-60 leukemic cells induced by VEGF.DESIGN:Repetitive measurement and observation.SETTING:School of Medicine, Wuhan University of Science and Technology.MATERIALS:The experiment was carried out in the Molecular Biology Laboratory Center, School of Medicine, Wuhan University of Science and Technology from March to June in 2007. Human leukemic cell line HL-60 cells were purchased from Shanghai Institute of Cell Biology. TMP hydrochloride injection was produced by Wuxi Seventh Pharmaceutical Products Limited (Lot number:011014), protamine sulfate injection was produced by Shanghai First Biochemical Pharmaceuticals (Batch number:010302), and immunohistochemistry kit was purchased from Boster company.METHODS:①Human leukemic cell line HL-60 cells at log phase were used for the experiments. Cells were treated with 100 μg/L VEGF, and then TMP at final concentrations of 1.5, 15, 150 mg/L was added into culture medium. While the cells in medium without TMP were taken as blank control group, and the cells in medium with 20 mg/L protamine as positive control group. Meanwhile cells without treatment of VEGF were served as VEGF control group. After cells were incubated for 48 hours, the growth inhibiting rate of HL-60 cells was detected by MTT assay.②After HL-60 cells were treated with TMP at the final concentrations of 1.5, 15, 150 mg/L for 24 hours, the protein expression of VEGF in HL-60 cells was examined by SP immunohistochemistry.MAIN OUTCOME MEASURES:①Growth inhibiting rate of HL-60 cells.②Protein expression of VEGF.RESULTS:①Growth inhibiting rate of HL-60 cells:After HL-60 cells induced by VEGF were treated with 15 and 150 mg/L TMP, the absorbance value was significantly lower than that in VEGF control group (P < 0.05).②Protein expression of VEGF:After HL-60 cells were treated with TMP for 24 hours, the protein expression of VEGF was down-regulated with increasing TMP concentration in a dependent manner. Significant differences were observed in the protein expression of VEGF between cells treated by TMP and the controls (P < 0.01).CONCLUSION:shRNA, by targeting hTERT mRNA, has no noticeable influence on growth and proliferation of hMSCs, and might be safe for the somatic cells which are normal but do not express hTERT.

ObjectiveTo discuss the effect of surgical treatment of varicose vein on primary chronic venous insufficiency(PCVI) in the lower extremities.MethodsBetween August 2007 and August 2008,128PCVIpatients underwenthighligationof the greatsaphenousveinandendovenous electrocoagulation of the varix superficial vein. Spectrum Doppler ultrasound was used to measure the superficial femoral vein blood flow hemodynamic information beneath the first pair of valve including quiet breathing condition and the Valsalva action (reflux time、caliber、reflux velocity). Reflux index (RI) was used as guide line.ResultsClinical sympotoms improved in 54 out of 60 mild PCVI cases, in 29 of 45 moderate PCVI patients, while only in 6 out 23 cases in whom severe preoperative clinical symptom with PCVI was present.The effective rate respectively was 90％, 64％, and 30％.The mild reflux index significantly improved than that before operation ( t = 21. 484, P = 0. 000 ), the moderate reflux index improved than that before operation ( t = 2. 173, P = 0. 035 ), while the serious reflux index were not statistically improved than that before operation( t = 1. 888, P = 0. 078 ). In all cases reflux index improved after the surgery ( x2 = 8. 266,P = 0. 004).ConclusionsMinimally invasive surgical treatment of varicose veins can improve the reflux of the deep vein in PCVI cases with mild to moderate clinical symptom.

Objective To prospectively assess the predictive power of centralization phenomenon in the curative effect of automated PLD.Methods The survey population was consisted of 109 patients with inclusion heraiation demonstrated by CT/MRI,74 men and 35 women with average age of 43.1 years(17～75 years). All were complained of low back pain,with varying degrees of lower extremity pain and altered sensation, lasting for more than 2 months;including one symptomatic disc in 99 patients and two symptomatic discs in 10 patients.Patients were undergone dynamic mechanical spinal test and reported whether the test would aggravate their pain.The assessment included forward flexion,extension,rotation of the trunk to the right and left, rotation to the left with fight extension,rotation to the fight with left extension,and whether straight leg raising in the supine position would aggravate back pain or leg pain.Symptom resposes were categorized into three groups:centralization group(CG),partial-centralization group(PCG)and noncentralization group(NCG). Centralization of pain is the progressive retreat of the most distal extent of the referred or radicular pain toward or to the lumbar midline.Noncentralization of pain is the peripheralization of pain in one or more directions, and no change in the distal-most pain location or intensity.All patients received a single therapy with PLD. Results A follow-up of 109 patients for 3 to 6 months,including 46 cases with 24 as exellent and 22 as good reaching 100% of excellent good rate in CG by MacNab standards;43 cases with 5 as exellent,29 as good,9 as fair and poor,with total effective rate of 79.1% in PCG.Twenty cases of NCG symptoms showed no improvement and therefore surgery was considered.Conclusions Centralization phenomenon occurrence during initial mechanical evaluation is a very accurate predictor for successful PLD outcome.Nonoccurrence of centralization would accurately predict poor PLD outcome and thus helpful as early predictor of the need for surgical treatment.

OBJECTIVETo analyze the effects of three surgical operations in the treatment of Pilon fracture of Rüedi-Allgower type III, and put forward the best therapeutic method.

METHODSThe clinical data of 33 patients with Pilon fracture who received surgical operations (plaster immobilization group, 10 cases; distal tibia anatomical plate group, 11 cases; external fixation with limited internal fixation group, 12 cases) from October 2009 to January 2012 were analyzed. There were 5 males and 5 females, ranging in age from 24 to 61 years in the plaster immobilization group. There were 7 males and 4 females, ranging in age from 21 to 64 years in the distal tibia anatomical plate group. There were 7 males and 5 females, ranging in age from 23 to 67 years in the external fixation with limited internal fixation group. The Ankle X-ray of Pilon fracture after operation, ankle score, early and late complications were collected. Bourne system was used to evaluate ankle joint function.

RESULTSAfter 8 months to 3 years follow-up, it was found that three kinds of treatment had significant differences in the outcomes and complications (P < 0.05): the external fixation with limited internal fixation group got the best results. The number of anatomic reduction cases in the external fixation with limited internal fixation group (7 cases) and the distal tibia anatomical plate group (8 cases) was more than the plaster immobilization group (2 cases). According to the ankle score, 8 patients got an excellent result, 3 good and 1 poor in the limited internal fixation group ,which was better than those of distal tibia anatomical plate group (5 excellent, 4 good and 2 poor) and the plaster immobilization group (3 excellent, 4 good and 3 poor). The number of early and late complications in the external fixation with limited internal fixation group was more than those in the plaster immobilization group and the distal tibia anatomical plate group (P< 0.05).

CONCLUSIONTreatment of external fixation with limited internal fixation in the treatment of Pilon fracture of Rüedi-Allgower type III is effective and safe.

Adult ; Aged ; Case-Control Studies ; Female ; Fracture Fixation, Internal ; methods ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Tibial Fractures ; diagnostic imaging ; surgery ; Tomography, X-Ray Computed ; Treatment Outcome ; Young Adult

OBJECTIVETo observe the protective effects of histone deacetylase inhibitor on stress-induced myocardial injury.

METHODSHealthy male Wistar rats were randomly divided into 3 groups( n = 6), and the stress-induced myocardial injury model was established with chronic restraint stress method. The protective effects of histone deacetylase inhibitor on stress-induced myocardial injury were observed with Trichostatin A (TSA) intervention. Histone acetylation levels in myocardium of rats were detected by Western blot method, spectrophotometry method was used to dynamically determine the activity of rat serum lactate dehydrogenase (LDH), serum creatine kinase isoenzyme-MB (CK-MB) and Caspase 3, and nagar Olsen staining were used to observe the early myocardial damage.

RESULTSRestraint stress could significantly reduce the level of histone acetylation of myocardium in rats, and TSA intervention could inhibit the stress-induced reduction of myocardial levels of histone acetylation. Restraint stress could cause the significant increase of serum LDH activity ( P < 0.05), serum CK-MB activity ( P < 0.05), and the Caspase 3 activity of myocardial tissue (P < 0.05), and early myocardial damage also occurred during restraint stress. ISA intervention could significantly reduce the serum LDH activity (P < 0.05), the serum CK-MB activity (P < 0.05), the activity of myocardial tissue caspase 3 induced by restraint stress (P < 0.05), and the stress-induced myocardial injury was also attenuated by TSA intervention.

CONCLUSIONThe histone deacetylase inhibitor TSA can protect stress-induced myocardial injury.

Acetylation ; Animals ; Cardiotonic Agents ; pharmacology ; Caspase 3 ; blood ; Creatine Kinase, MB Form ; blood ; Histone Deacetylase Inhibitors ; pharmacology ; Hydroxamic Acids ; pharmacology ; L-Lactate Dehydrogenase ; blood ; Male ; Myocardium ; pathology ; Rats ; Rats, Wistar ; Restraint, Physical ; Stress, Physiological

A liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was developed for the determination of budesonide in dog plasma. Budesonide and the internal standard triamcinolone acetonide were separated from plasma by alkalinized liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a Capcell Pak C18 MG column with the mobile phase consisted of acetonitrile -5 mmol x L(-1) ammonium acetate (60:40, v/v) at a flow-rate of 0.50 mL x min(-1). A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the negative ion mode. Quantification was performed using multiple reaction monitoring (MRM) of the transitions m/z 489 --> m/z 357 and m/z 493 --> m/z 413 for budesonide and the internal standard, respectively. The linear calibration curves were obtained in the concentration range of 25.0-2000 pg x mL(-1). The lower limit of quantification was 25.0 pg x mL(-1). The intra- and inter-day relative standard deviation over the entire concentration range was less than 15%. The accuracy was in the range of -8.1% to -1.7% in terms of relative error. The method was applied to a pharmacokinetic study of budesonide controlled-release capsules in Beagle dogs. Maximal budesonide plasma level was observed after (3.5 +/- 3.3) h and the Cmax was (786 +/- 498) pg x mL(-1) after a single oral administration of 9 mg budesonide capsules, Cmax was increased to (2142 +/- 1515) pg x mL(-1) after multiple oral administration (9 mg x 5 d) of budesonide capsules. This method was selective and rapid, and the sensitivity was sufficient for the purpose of the pharmacokinetic study of budesonide controlled-release formulation.
Animals ; Anti-Inflammatory Agents ; blood ; pharmacokinetics ; Area Under Curve ; Budesonide ; blood ; pharmacokinetics ; Chromatography, Liquid ; methods ; Delayed-Action Preparations ; Dogs ; Male ; Sensitivity and Specificity ; Spectrometry, Mass, Electrospray Ionization ; Tandem Mass Spectrometry ; methods

AIMTo develop and validate a liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the simultaneous quantification of ephedrine and chlorpheniramine in human plasma after oral administration of a compound preparation.

METHODSThe analytes and the internal standard, diphenhydramine, were isolated from plasma by protein precipitation with methanol, then chromatographied on a Zorbax SB-C18 column (150 mm x 4.6 mm ID) using a mobile phase consisted of methanol-water-formic acid (80: 20: 0.5, v/v), at a flow rate of 0.5 mL x min(-1). A tandem mass spectrometer equipped with electrospray ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor to produce ion combinations of m/z 166-->115, m/z 275-->230 and m/z 256-->167 were used to quantify ephedrine, chlorpheniramine and the internal standard, respectively. Results The linear concentration ranges of the calibration curves for ephedrine and chlorpheniramine were 0.50 - 200 microg x L(-1) and 0.050 - 20.0 microg x L(-1), respectively. The lower limits of quantification were 0. 50 microg x L(-1) for ephedrine and 0.050 microg x L(-1) for chlorpheniramine, individually. The intra- and inter-day relative standard deviation (RSD) across three validation runs over the entire concentration range was less than 9.3% for both ephedrine and chlorpheniramine. The inter-day accuracy (RE) was within +/- 3.4% for the analytes. Each sample was chromatographied within 3.3 min. The method was successfully used in pharmacokinetics study of ephedrine and chlorpheniramine in human plasma after oral administration of a compound preparation containing 5 mg ephedrine hydrochloride, 1 mg chlorpheniramine maleate, 50 mg phenytoin, 12.5 mg theophylline, 12.5 mg theobromine and 7.5 mg caffeine. No interaction among the six components was observed on their pharmacokinetic parameters.

CONCLUSIONThe method was proved to be highly sensitive, selective, and suitable for pharmacokinetics investigations of different compound preparations containing low dosage of both ephedrine and chlorpheniramine.

Administration, Oral ; Area Under Curve ; Chlorpheniramine ; administration & dosage ; blood ; pharmacokinetics ; Chromatography, Liquid ; methods ; Drug Combinations ; Ephedrine ; administration & dosage ; blood ; pharmacokinetics ; Humans ; Male ; Spectrometry, Mass, Electrospray Ionization ; methods

AIMTo develop and validate a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the determination of risperidone in human plasma.

METHODSRisperidone and the internal standard, diphenhydramine, were isolated from plasma by liquid-liquid extraction with etherdichloromethane (3:2, v/v) , then chromatographed on a Zorbax Extend-C18 column (150 mm x 4.6 mm ID, 5 microm) using a mobile phase consisted of acetonitrile-water-formic acid (40:60: 0.5, v/v), at a flow rate of 0.7 mL x min(-1). A Finnigan TSQ tandem mass spectrometer equipped with atmospheric pressure chemical ionization source was used as detector and was operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor product ion combinations of m/z 411-->191 and m/z 256-->167 were used to quantify risperidone and diphenhydramine (IS) , respectively.

RESULTSThe linear concentration range of the calibration curve for risperidone was 0.025 - 50 microg L(-1). The lower limit of quantification was 0.025 microg x L(-1). The intra- and inter-day relative standard deviation (RSD) across three validation running over the entire concentration range was less than 7.1%. The accuracy was within +/- 3.8%. Each sample was chromatographed within 2.7 min.

CONCLUSIONThe method was proved to be rapid, sensitive and suitable for pharmacokinetic investigations of risperidone.

Antipsychotic Agents ; administration & dosage ; blood ; pharmacokinetics ; Chromatography, Liquid ; methods ; Diphenhydramine ; blood ; standards ; Humans ; Male ; Reference Standards ; Reproducibility of Results ; Risperidone ; administration & dosage ; blood ; pharmacokinetics ; Tandem Mass Spectrometry ; methods

OBJECTIVETo study the passive immunization with human monoclonal antibodies as for prophylaxis of human cytomegalovirus (HCMV) infection.

METHODSFab monoclonal antibodies to HCMV were recovered by repertoire cloning of mRNA from a HCMV infected individual. Antigen binding specificity, CDR sequence of V(H) and V(L) and neutralizing activity on HCMV AD169 stain were analyzed in vitro. The light and heavy chain Fd fragment genes of Fab antibodies were further cloned into a recombinant baculovirus expression vector pAC-kappa-Fc to express intact IgG. Secreted products were purified with affinity chromatography using protein G.

RESULTSSDS-PAGE and Western blot confirmed the expression of the intact IgG. Immuno-blotting and -precipitation were used to identify HCMV proteins. One Fab monoclonal antibody recognized a conformational HCMV protein.

CONCLUSIONIgG antibodies can neutralize the HCMV AD169 strain efficiently at a titer of 2.5 microg/mL and may prove valuable for passive immunoprophylaxis against HCMV infection in humans.

Amino Acid Sequence ; Animals ; Base Sequence ; Cell Line ; Cloning, Molecular ; Cytomegalovirus ; immunology ; DNA, Viral ; genetics ; Genes, Viral ; Humans ; Immunoblotting ; Immunoglobulin G ; immunology ; Immunoprecipitation ; Insecta ; Molecular Sequence Data ; Peptide Library ; Recombinant Proteins ; immunology