The objective of this study was to screen out the effective shRNA which can inhibit the gene expression of tumour necrosis factor-alpha (TNF-alpha), to construct the recombinant plasmid and to determine its sequence so as to provide the new approach for searching gene therapy of TNF-alpha related diseases. The primary macrophages were added into 15% DMEM, then cells were adjusted as 2 x 10(7) cells/L and were inoculated in 6-well plate with 3 ml/well, and were cultured at 37 degrees C in a fully humidified atmosphere with 5% CO(2). Cells were stimulated with lipopolysaccharide (LPS) and the concentration of TNF-alpha in the supernatant at different time points was determined by enzyme-linked immunosorbent assay (ELISA). The 5 synthesized DNA sequences which can be transcripted into shRNA were transfected into cells with lipofectamine 2000, then the cells were stimulated with LPS for 24 hours. The concentration of TNF-alpha in the supernatant and the expression of TNF-alpha mRNA were determined by ELISA and reverse transcription polymerase chain reaction (RT-PCR) respectively. The most effective shRNA was inserted into plasmid, and the recombinant plasmid was identified by sequence analysis. The results showed that the concentration of TNF-alpha in the supernatant reached peak after the stimulation with LPS for 24 hours. In the RNA interference group, the shRNA 1 was the most effective one, which could inhibit the expression of TNF-alpha by 59.46% and the expression of TNF-alpha mRNA by 61.2%. The recombinant plasmid was cloned and the sequence of interest was obtained. In conclusion, the most effective shRNA targeting TNF-alpha was successfully screened out and the recombinant plasmid was constructed. The recombinant plasmid may be helpful to search new gene therapy for TNF-alpha related disease.
Animals ; Cells, Cultured ; Gene Expression ; Genetic Therapy ; Genetic Vectors ; Macrophages ; cytology ; Male ; Mice ; Mice, Inbred C57BL ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Recombination, Genetic ; Transfection ; Tumor Necrosis Factor-alpha ; genetics

Objective To evaluate the effectiveness of management for patients with chronic obstructive pulmonary disease (COPD) in stage Ⅰ at new rural cooperative medical facilities.Methods A quasi-experiment design was used for the evaluation of patients registering at Dongcun and Fangyuan subdistrict offices in Haiyang.And 79 COPD cases from Dongcun sub-district offices were selected into the management group while 76 cases from Fangyuan the control group.The measures at the new rural cooperative medical facilities included smoking-free publicity,avoiding a hazardous environment,application of influenza vaccines and training of patients and local general practitioners in essences of COPD prevention and treatment.Except for routine treatment,no special measures were taken for the control group.ResultsSixty-one patients in the management group and 60 in the control group completed a 3-year follow-up.Fifteen patients were smokers during their initial visits in the management group and 13 quitted smoking after a 3-year management.Meanwhile 19 patients were smokers during their initial visits in the control group and 3 of them quitted smoking within the same period.The decrement of BODE index between post-management at year 2&3 and between pre-management was higher in the management group versus the control group within the same period (P ＜ 0.01).The annual average frequencies of common cold,acute attack and physician consultation were obviously lower post-management at year 3 than those pre-management and the control group within the same period ( P ＜ 0.05 ).Lung functions of two groups deteriorated as compared with those 3 years before ( P ＜ 0.05).But no statistical difference existed between two groups (P ＞ 0.05).ConclusionThe management of COPD patients in stage Ⅰ at new rural cooperative medical facilities can improve their quality of life and reduce their frequencies of common cold,acute attack and physician consultation.

Objective To build the cohort of drug resistance and analyze treatment efficiency of AIDS patients and situation of drug resistant mutations among HIV-1 infected individuals.Methods A cohort of 116 HIV-1 infected patients was built and their treatment progress were acquired once every 6 months.At the sanle time CD4+ T cell counts and HIV-1 viral load were measured and genotyping for drug resistance was determined by a home brew nested PCR.Results The CD4+ T cell count(470±251/ml)was higher than that before treatment in patients who were treated by AZT/DDI/NVP or D4T/DDL/NVP.The viral load was lower than that before treatmenL The drug resistant mutation frequency increased gradually along with treatment.The CD4+ T cell count was decreased and viral load was increased and the prevalence of drug resistant mutation was increased in the patients who changed regimens to AZT/3TC/NVP or D41/3TC/NVP.Only one primary mutation that was resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs)was detected in the naive patients.The cross-resistant mutation was detected in two patients after 6 months treatment. The intermediate resistance to lopinavir(LPV) was detected after 12 months treatment.The prevalence of high-grade resistances to NNRTIs was increased obviously,and the prevalence of multi-resistance and cross-resistance was detected in 5 patients after 36 months treatment.Conclusions The prevalence of primary mutation was rare in naive HIV-1 infected patients.The prevalence of drug resistant mutation was inereased gradually along with treatment.Ahhough few regimens were available,the treatment effect could last relatively long period of time if patients keep taking medicine stably.The regimens could be changed according to the results of drug resistant test.

OBJECTIVETo investigate the antithrombotic effects and its molecular mechanisms of prazosin combined with anisodamine (Ani).

METHODSIsolated rat tail artery rings model was employed to evaluate the vasodilative effects of drugs, mice tail thrombosis model induced by carrageenan was used to study the antithrombotic effects and its molecular mechanisms of the drug composition.

RESULTSAmong alpha1-adrenoreceptor antagonists, prazosin(Pra) had the greatest relaxation rate, which was (82.6 +/- 8.9)%, and the EC50 value was 0.44 micromol/L. The drug composition of anisodamine and prazosin of different doses could decrease the length of the tail thrombosis from (24.6 +/- 4.6)mm to (6.9 +/- 2.7)mm, and the rate of thrombosis was decreased from 86.6% to 50.0%. The drug composition could prolong the prothrombin time (PT) distinctively, but it had no effect on the activated partial thromboplastin time (APTT). It also could restrain the decrease of serum levels of tissue plasminogen activator (t-PA) and 6- Keto -PGF1alpha as well as the increase of type-1 plasminogen activator inhibitor (PAI-1) and thromboxane B2 (TXB2) in the mice.

CONCLUSIONThe drug composition formed by anisodamine and prazosin has good effects of relaxing extremities tiny blood vessels and it can fight against thrombosis, its antithrombotic mechanisms may be related to the influence of the extrinsic coagulation pathway, inhibition of platelet activation functions and the promotion of fibrinolysis function.

Adrenergic alpha-1 Receptor Antagonists ; pharmacology ; Animals ; Disease Models, Animal ; In Vitro Techniques ; Male ; Prazosin ; pharmacology ; Rats ; Rats, Wistar ; Solanaceous Alkaloids ; pharmacology ; Thrombosis ; drug therapy

Objective To study the inhibitory effects of TPP,a desmosdumotin-C B ring para-fluoro modified derivative,on human breast cancer MCF-7 cell proliferation,and investigate the possible mechanisms.Methods MTT assay was used to measure the proliferation suppression of MCF-7 cells after treated with 1.0,2.5,5.0,10.0,and 20.0 μg/mL TPP for 48 h,and then the cell apoptosis rate and expression rate of NF-κB P65 positive cells were tested by flow cytometry after 20.0 μg/mL TPP treatment for 0,24,and 48 h.Results MTT assay showed that,after treatment for 48 h,1.0,2.5,5.0,10.0,and 20.0 μg/mL TPP all exhibited the inhibitory effects and showed a dose-dependent relationship.Flow cytometry results showed that 20.0 μg/mL TPP induced cell apoptosis after treatment for 24 and 48 h.TPP (20.0 μg/mL) significantly reduced the rate ofNF-κB P65-positive cells in MCF-7 cells after treatment for 48 h.Conclusion TPP could inhibit the proliferation of MCF-7 cells,which may be induced by cell apoptosis.Down-regulation of NF-κB is possible to be related with apoptosis.

Objective To evaluate the prognostic impact of MRI-detected prevertebral space involvement in nasopharyngeal carcinoma (NPC) treated with radiotherapy and chemotherapy.Methods A retrospective analysis was performed on the clinical data of 333 patients who had newly diagnosed biopsyproven NPC without distant metastasis from 2005 to 2007.All patients underwent MRI scans of the nasopharynx and neck and were treated with two-and three-dimensional radiotherapy without or without chemotherapy.The Kaplan-Meier method was used to calculate overall survival (OS),distant metastasis-free survival (DMFS),and locoregional relapse-free survival (LRFS),and the log-rank test was used for survival difference analysis;the Cox proportional hazards regression analysis was used to assess the prognostic value of prevertebral space involvement.Results The follow-up rate was 95.2％.Prevertebral space involvement was seen in 139(41.7％) of these patients.The patients with prevertebral space involvement had significantly higher T stage and clinical stage than those without prevertebral space involvement (x2 =90.41,P =0.000;x2 =54.03,P =0.000).The 5-year OS,DMFS,and LRFS for NPC patients with and without prevertebral space involvement were 58.8％ vs.77.5％ (x2 =11.95,P =0.000),77.8％ vs.85.0％(x2=2.56,P=0.110),and 88.3％ vs.91.8％ (x2=1.51,P=0.220),respectively.After adjusting for N stage,a significant difference was still seen between the two groups with regard to 5-year OS (x2 =9.93,P =0.002).The multivariate analysis showed that prevertebral space involvement was not the independent prognostic factor for OS,DMFS,and LRFS (x2 =0.43,P =0.512 ; x2 =0.08,P =0.783 ; x2 =0.00,P =0.971).Conclusions The frequency of prevertebral space involvement is very high in NPC.The OS for the patients with prevertebral space involvement is significantly lower than those without prevertebral space involvement.But prevertebral space involvement is not the independent prognostic factor in NPC patients.

Liquid chip technology have been licensed to be used in clinic because of its advantage of high-throughput, high-sensitivity, good signal to noise ratio, reaction in liquid phase, convenient operation and short time consuming, etc. The optimization of a liquid chip system for the detection of serum biomarkers of colorectal tumour and initial application in the detection of CEA were studied. The optimized reaction conditions of liquid chip were determined through orthogonal design after it was prepared. The results showed that the consuming reaction time of the coated antibody and the antigen was 1hour. The microspheres, biotinylated detecion antibody and the consuming complexes and avidin-PE time of the microspheres and the biotinylated tested antibody was 1hour, 1hour and 15minutes respectively.the consuming time of the complexes and avidin-PE was fifteen minutes, The optimized dilution of the biotinylated tested detection antibody was 1∶300 and the optimized concentration of avidin-PE was 12?g/ml. Totally 55 clinical samples were detected by the liquid chip and by Enzyme-Linked Immunosorbent Assay (ELISA) simultaneously and the results of the two methods were compared. The results of the two methods showed good correlation between positive and negative samples but the detection limits and the dynamic ranges of the liquid chip method were more sensitive and wider than those of the ELISA. The multiple tumour biomarkers may be detected simultaneously and the time of clinical test and manpower requirements were reduced by the liquid chip method.

By non-steady method,the effective diffusivity of ferrous sulphate within alginate calcium gel entrapped without bacteria was measured.Meanwhile the oxidation ability of entrapped bacteria was analyzed.Experimental results showed that the effective diffusion coefficient of ferrous sulphate decreased with the increase of alginate concentration,the optimum alginate concentration is 2%(W/V).The effect of calcium chloride on the effective diffusivity was neglectable.The incubation of ferrooxidans would pass through 10 hours,and the diffusion coefficient within gel entrapped Thiobacillus ferrooxidans cells was less remarkably than that of ferrous sulphate without entrapped cells.For the entrapped cells,the absolute oxidation time was shortest and the rate change was fastest with the initial Fe concentration 5g/L.The absolute oxidation time was same when the initial Fe concentration was 8g/L and 10g/L.

Objective To investigate the prognostic value of maximum diameter in axial plane of primary tumor (MDAPPT) on MRI in nasopharyngeal carcinoma (NPC).Methods From 2005 to 2007,333 patients with newly diagnosed and biopsy-proven NPC without distant metastasis,who underwent MRI scans of the nasopharynx and neck,were included in our study.MDAPPT was measured on MRI.The univariate analysis with the log-rank test and multivariate analysis with the Cox proportional hazards model were used to analyze the relationship between MDAPPT and prognosis.Results The median values of MDAPPT in patients with T1,T2,T3,and T4 NPC were 21.2,30.0,38.0,and 52.3 mm,respectively.For all patients with a MDAPPT of ≤30 mm,＞ 30-50 mm,and ＞ 50 mm,the 5-year overall survival (OS) rates were 81.3％,70.1％,and 51.5％,respectively (P =0.000) ; the 5-year progression-free survival (PFS) rates were 81.3％,70.0％,and 48.9％,respectively (P =0.000) ;the 5-year distant metastasisfree survival (DMFS) rates were 85.5 ％,86.5 ％,and 67.2 ％,respectively (P =0.000) ; the 5-year local relapse-free survival (LRFS) rates were 97.7％,91.5％,and 83.3％,respectively (P =0.013).The multivariate analysis showed that MDAPPT was a prognostic factor for PFS and DMFS.For the T3-T4 patients with a MDAPPT of ≤50 mm and ＞50 mm,the 5-year OS rates were 69.4％ and 52.2％ (P =0.004),the 5-year PFS rates were 68.0％ and 49.6％ (P =0.001),and the 5-year DMFS rates were 84.0％ and 66.8％ (P=0.001).In the patients with a MDAPPT ≤30 mm,the 5-year LRFS rates for those with T1,T2,T3,and T4 NPC were 10 0 ％,9 5.8 ％,9 6.3 ％,and 10 0 ％,respectively (P =0.6 4 3).Conclusions MDAPPT is a prognostic factor for PFS and DMFS in NPC,and it is an important prognostic factor in patients with T3-T4 NPC.In the NPC patients with a small MDAPPT,local control rate varies little in different T stages.

Objective To investigate the feasibility of using low Kv,low iodine contrast Agent concentration (dual low)CT scan techniques in Coronary CTA (CCTA).Methods Seventy-six patients undergoing CCTA were divided into Group A and Group B , randomly.Group A (38 patients)was the dual-low group,which was scanned with tube voltage of 100 kVp,and injected with iso-osmolarity contrast agent visipaque 270 (270 mg I/mL),with iterative reconstruction technique (ASIR 40%).Group B (38 pa-tients)was scanned with 120 kVp,and low osmolarity contrast agent omnipaque 350 (350 mg I/mL)and FBP reconstruction,The images are assessed double-blindly by two experienced radiologists.Five ROIs were placed onto the ascending root of aorta (AO), left main artery(LM),left anterior descending (LAD),left circumflex artery(LCX),right coronary artery (RCA),and the image qualities are evaluated objectively using CT values,noise,signal noise ratio (SNR),contrast noise ratio (CNR),and compared sta-tistically using Paired t-test.The radiation dosages,such as CTDIvol,DLP and ED were also recorded and compared with Paired t-test.Results CTDIvol,DLP and ED of Group A (dual low)decreased 35.7%,38.6% and 38.6% respectively compared with Group B,the iodine intake decreased 22.9%.While the image qualities of the two groups were not significantly different,all images are good enough for diagnosis,with Group A slightly better than Group B in radiologists’scores.Conclusion Voltage 100 kVp, combined with low contrast agent concentration of 270 mg I/mL can fully satisfy the diagnostics need in CCTA,and significantly lower both the radiation dosage and iodine intake.