The aim of this study is to clone the mouse T-bet promoter and enhancer, construct and identify the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS for T-bet transcription regulation study and its function in signaling of multiple sclerosis. The promoter and CNS of T-bet were predicted by bioinformatics assay. The predicted fragment of mouse T-bet promoter plus CNS was amplified by PCR and cloned into pGL4.10. The recombinant plasmid pGL4.10-TBX21pr-CNS was transferred into Escherichia coli DH5α. The positive clone was identified by double digestion with Kpn I and Sfi I and DNA sequencing. Finally, pGL4.10-TBX21pr-CNS was cotransfected with pRL-TK into 293T cells and Jurkat cells, pRL-TK and pGL4.10 as a control. The luciferase activity in 293T cells (P = 0.012 2) and Jurkat cells (P = 0.002 2) was higher than that of the control group. A fragment of 1 028 bp mouse T-bet promoter plus 1 308 bp CNS was successfully cloned and the firefly luciferase reporter gene plasmid pGL4.10-TBX21pr-CNS was constructed. In 293T cells and Jurkat cells, pGL4.10-TBX21pr-CNS has the promoter functions. This work offers a basic material for the research of T-bet transcription.
Animals ; Gene Expression Regulation ; Genes, Reporter ; Genetic Vectors ; Luciferases ; Mice ; Multiple Sclerosis ; Plasmids ; Promoter Regions, Genetic ; T-Box Domain Proteins ; genetics

Objective To observe the impacts of tirofiban on plasma levels of sCD40L and HIF-1α, and iNOS activity in patients undergoing emergency PCI. Methods A total of 80 patients with acute STEMI received primary PCI were randomly divided into a tirofiban group and a control group. The tirofiban group received tirofiban of 10 μg/kg, followed by an infusion of tirofiban of 0.15 μg/(kg·min) for 24 ~ 36 hours; while the control group directly received stent implantation after balloon angioplasty. Plasma levels of sCD40L , HIF-1α, and iNOS were detected in baselines and 12 hours after the procedrue. Results 12 hours after the procedure, serum levels of sCD40L, HIF-1α, and iNOS were higher than the baseline levels (P < 0.05). As compared with the control group, levels of sCD40L, HIF-1α, and iNOS were significantly lower (P < 0.05). Conclusions This study preliminarily sugguests tirofiban has a role in anti-inflammation and protection of vascular endothelium besides anti-platelet effects.

Objective To establish a cell line with repressed expression of p53 by transfecting a plasmid construct expressing short hairpin RNA(shRNA)targeting p53 into human skin fibroblasts(HSFs),and to evaluate the effect of repression of p53 expression on the senescence in HSFs.Methods The eukaryotic expressing plasmid pGCsi-p53 containing shRNA targeting p53 gene was transfected into HSFs with lipofectamine.Subsequently,the cells were selected by G418,and resistant cell clones were chosen and expanded.Reverse transcription-PCR and real time fluorescence-based quanitative PCR were performed to determine the expression of p53 gene,and Western blot to detect the expression of p53 protein in HSFs.The senescence in HSFs was evaluated by SA β-gal staining,and cell proliferation by methyl thiazolyl tetrazolium(MTT)assay.Results A HSF clone with repressed expression of p53 was established successfully.The expressions of p53 mRNA and protein were downregulated in transfected HSFs compared with untransfected HSFs(0.09 ± 0.03 vs.0.32 ± 0.04,0.11 ± 0.04 vs.0.84 ± 0.05,both P ＜ 0.01).The percentage of senescent cells was 13.47％ ± 1.01％ in the transfected HSFs,significantly lower than that in untransfected HSFs(18.10％ ± 0.66％,P ＜ 0.05).As MTT assay showed,the proliferation was accelerated in transfected HSFs compared with untransfected HSFs(P ＜ 0.05).Conclusions The repression of p53 expression decelerates the senescence in HSFs,but promotes the proliferation of HSFs.

Objective To discuss the efficacy of edaravone combined with batroxobin in the treatment of patients with acute progressive infarction,and its influence on neurological deficit and coagulation.Methods 64 patients with acute and progressive infarction were selected,they were randomly divided into the control group and the observation group,32 cases in each group.The patients of the observation group were given edaravone combined with batroxobin,while the patients of the control group were given batroxobin only.Results The total effective rate of the observation group was 91.63%,which was obviously higher than 78.13% of the control group(χ2 =11.274,P <0.05).The NDS of the observation group was (14.8 ±2.9 )points,which was obviously lower than (17.9 ± 3.3)points of the control group(t =9.98,P <0.05).The level of Fib of the observation group was (2.9 ±0.3)g/L, which was obviously lower than (3.2 ±0.4)g/L of the control group(t =9.34,P <0.05).Conclusion Edaravone combined with batroxobin in the treatment of patients with acute and progressive infarction has significant effect and deserve promotion.

Objective To explore the stress perception among acute myocardial infarction (AMI) patients during acute stage.Methods Phenomenological method of qualitative study was adopted.18 AMI patients during the acute stage of 3～4 days after the onset of the disease were interviewed.Results Stress made AMI patients hard to accept the fact of illness,worry about the treatment of illness and the impact on future life,be difficult to alter living habits,be hard to relieve negative emotion.Conclusions AMI patients during acute stage will experience different levels of psychological stress.Stress management should be fabricated and effective measures should be adopted to relieve their negative emotions in terms of inducing factors.

This paper aimed to study clinical effectiveness of gabapentin in the treatment for pruritus and insomnia among patients with hemodialysis. Eighty patients with severe pruritus and insomnia during regular hemedialysis in Jiaozhou People's Hospital, Jiangsu were randomly divided into two groups with 40 patients each. Gahapentin 0. 1 g were given each patient every night for 4 weeks in one group and a modified-re]ease capsule of 8 mg chiorphenamine maleate was given each patient daffy for 4 weeks in the other group. Effectiveness of treatment was evaluated by improvement of itching symptoms and Pittsburgh Sleep Quality Index (PSQI) scores for insomnia in the patients. Itching improvement was observed in 85%(34/40) of the patients and PSQI scores obviously lowered in them four weeks after gabapentin treatment.However, itching improvement was observed only in 42% ( 17/40 ) of the patients and little change was observed in PSQI scores in them four weeks after chlorphenamine treatment It suggests that gabapentin can significantly improve quality of sleeping and itching symptoms in patients with hemodialysis and it is worthy popularizing in those with it.

Objective To investigate the neural mechanism of inhibitory control in sensation seeking by using the event-related potential(ERP) technique. Methods High and low sensation seekers( 16 people in each group ), who were selected according to their sensation seeking scores, performed a Go/Nogo task in which the stimuli possessed two levels of difficulty. Electro- encephalogram(EEG) signals were recorded continuously by a set of 32 Ag/AgCI electrodes. Results For two types of stimuli ( Congruent, Incongruent) :( 1 )The amplitude(FCZ) of Nogo N2 and Nogo P3 were( (1.61 ±4.25)μV,(-2.32±4.55)μV)and((16.44±5.74)μV,(17.00±5.71)μV). (2)There was no significant main effects of group for the Nogo N2 amplitude( F (1.30) =0.31, P=0. 59,η2=0. 01;F(1.30) =0.07,P=0.80,η2=0.002) ,the N2d amplitude( F(1.30) =1.18,P=0.29,η2=0.04;F(1.30) =0.004, P=0.95, η2 ＜ 0.001 ) ,the Nogo P3 amplitude( F (1.30) =0.13, P=0.72, η2 =0.004;F(1.30)=0.28, P=0.60, η2 =0.009) and the P3d amplitude( F(1.30) =0.08, P=0.50, η2 =0.02; F (1.30) =0.56,P=0.46, η2 =0.02). (3)Neither of main effects for the N2 and P3 latency was significant (P＞0.05). Conclusion The inhibitory control is similar across high and low sensation seeking groups,indicating that there is no relationship between the sensation seeking behaviors and the individual inhibitory control.

0 ^05). Giemsa positive samples were all positive by PCR-ELISA. The negative control group had one positive by ELISA in lung tissue and BALF respectively. Conclusion PCR-ELISA shows a high sensitivity and specificity in detecting the DNA of Pneumocystis carinii, which is a secure and easy use method.

Background Oxidative stress is a pathophysiological process of retina,so it is very important to explore a protective way against retinal oxidative stress.Studies determined that extract of ginkgo biloba (EGb) has antioxidant,anti-apoptosis,anti-thrombosis and anti-inflammatory effects,however,the effect of EGb on human Müller cells in oxidative stress is still below understood.Objective This study was to investigate the protection of EGb against oxidative stress of human retinal Müller cells induced by As2O3 in vitro.Methods Human retinal Müllercell line was cultured in DMEM/F12 with 10％ fetal bovine serum (FBS),2 μmol/L glutamine and 1％ antibiotics.As2O3 solution at the final concentration 5 mg/L was added in the medium for 24 hours to establish oxidative models,and then the EGb with the final concentrations of 5,10 and 20 mg/L was used to cell models for 24 hours,respectively.Cell viability was detected by MTT assay,and reactive oxygen species (ROS) levels in the cells were detected with CM-H2DCFDA fluorescent probe.The relative expression levels of caspase-3 mRNA in cytoplasm and cell nuclei were assayed by reverse-transcription PCR (RT-PCR),and the expressions of Nrf2 protein were quantitatively detected by Western blot.Results Müller cells adhered well 24 hours after cultured.At 6-7 days after culture,Müller cell body was large with abundant cytoplasm and mosaic-like arrangement.However,floating cells were seen after As2 O3 treatment.Cell viability (absorbance) was significantly different among the normal culture group,As2 O3-treated group,As2 O3 + 5 mg/L EGb group,As2 O3 + 10 mg/L EGb group and As2 O3 + 20 mg/L EGb group,with the strongest viability in the normal culture group and the weakest viability in the As2 O3-treated groups (F=163.57,P =0.00).The fluorescence intensity of ROS was the weakest in the normal culture group and the strongest in the As2 O3-treated group and was gradually weakened with the increase of EGb doses,showing a remarkable difference among the groups (F =4 013.61,P =0.00).The relative expression level of caspase-3 mRNA in the cells was gradually reduced with the increase of EGb doses,with a statistically significant difference among the groups (F =2 199.72,P =0.00).In addition,no considerable difference was seen in the expression level of Nrf2 protein (grey scale) in cytoplasm among the groups (F=15.42,P=0.40);while in the nuclei,the expression levels of Nrf2 protein were 100.01 ±0.04,46.59±0.63,54.51 ±0.62,59.93 ±0.17 and 67.60±0.24 in the normal culture group,As2 O3-treated group and As2O3+5 mg/L EGb group,As2O3+10 mg/L EGb group,As2O3+20 mg/L EGb group respectively,with a significant difference among them (F=7 271.72,P=0.00).Conclusions EGb can protect human retinal Müller cells against As2O3-induced damage in a dose-dependant manner by antioxidant and antiapoptotic effects in vitro,and the activities occur primarily in cell nucleus.

Objective To investigate the relative activity of multidrug resistance P-glycoprotein in the plasma of breast cancer patients, analyze the expression of P-glycoprotein before and after chemotherapy and evaluate its clinical value. Methods The plasma of patients and chemical reagents were mixed and distributed into the 96-well plate. The plate was read at 340 nm absorbaace before and after reaction. The variation of absorbance for each well was calculated, and then relative activity of each sample was compared to the referent compound activity. The relative activity of over 30 % was taken as positive expression of the P-gp for each sample. Results The frequency of P-glycoprotein expression was significantly increased from 16.2 % before chemotherapy to 43.2 % after chemotherapy (P<0.05). The positive expression of P-glycoprotein before and after chemotherapy were 5/6 and 9/16, respectively, in patients with positive lymph node metastasis, and 1/6 and 7/16, respectively, in patients with negative lymph node metastasis. Conclusion The results of this preliminary study suggest in P-glycoprotein expression in plasma of patients play an important role in the selection of drugs, which may be useful to evaluate the prognosis for treatment of breast cancer patients.