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 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.

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.

Abstract: Neurotensin receptor-1 (NTR1), which can stimulate the intracellular cascade signal pathway, belongs to the large superfamily of G-protein coupled receptors. NTR1 is related to the occurrence and development of several kinds of diseases. In order to screen the inhibitors for the cancers associated with NTR1 protein, we established a CHO (Chinese hamster ovary) cell line in which human neurotensin receptor-1 was highly expressed. The method is to construct the recombinant plasmid which was lysed with the hNTR1 gene and transfect it into CHO cells. After selected with G418, the cell line was evaluated by Western blotting analysis and calcium flux assays. Through the calcium flux assays on FlexStation 3, we got the EC50 value of neurotensin peptide which is the natural NTR1 agonist, and the IC 50 value of SR48692 which is the known NTR1 antagonist. The established human CHO/NTR1 cell line can be used to study the profile of NTR1 biological activity and further screen of NTR1 antagonists and agonists.

Background and purpose:DNA methylation is a common epigenetic alteration in cervical carcino-genesis. The aim of this study was to measure the levels of LMX1A and PAX1 gene methylation in cervical cancer and pre-cursors and to identify their potential in clinical application. Methods:Cervical specimens were collected from 121 female patients including 27 cases with invasive cervical cancers (ICC), 34 cases with high-grade cervical intraepithelial neoplasia (HG-CIN), 32 cases with low-grade cervical intraepithelial neoplasia (LG-CIN) and 28 cases with chronic cervicitis as normal controls (NLM). DNA methylations of the LMX1A and PAX1 gene were quantified using the techniques of nest PCR and pyrosequencing. The mean methylation values of the 6 gene loci on the LMX1A gene and the 9 gene loci on the PAX1 gene were respectively calculated for a given sample. Receiver operating characteristic (ROC) curve was used to evaluate the accuracy of gene methylation analysis to discriminate the cervical diseases. Results:The mean methylation value of the LMX1A gene in ICC was 14.36%, which was significantly higher than those in HG-CIN (4.70%), LG-CIN (5.05%) and NLM (4.53%) (P<0.01). The mean methylation value of the PAX1 gene in ICC was 41.97%, which was significantly higher than those in HG-CIN (10.21%), LG-CIN (5.55%) and NLM (4.92%) (P<0.01). The area under the ROC curve (AUC) was 0.603 for LMX1A gene methylation, and was 0.883 for PAX1 gene methylation, to discriminate ICC from HG-CIN, LG-CIN, and NLM (P=0.104 and<0.001, respectively). The optimal cut-off value for PAX1 gene methylation was set at 20.50%with the sensitivity of 81%and with the specificity of 93%. If the cut-off value was set at 9.58%, the sensitivity and the specificity of PAX1 gene methylation were 89%and 84%respectively. Conclusion:Quantitative analysis of the PAX1 gene methylation in cervical tissue might be helpful to differentiate invasive cancers from precursors, while the clinical applica-tion of the LMX1A gene methylation was limited.

Objective To evaluate the effect of RNA interference in p53 gene on the expressions of genes involved in ultraviolet B (UVB)-induced premature senescence and photocarcinogenesis in human skin fibroblasts (HSFs).Methods A previously established HSF cell clone with repressed expression of p53,which was named as HSF-p53,was cultured and irradiated with a subcytotoxic dose (10 mJ/cm2) of UVB once a day for five consecutive days.The HSFs with normal expression of p53 served as the control.Subsequently,β-galactosidase (SA-β-gal)-staining was performed to estimate the degree of senescence,quantitative real-time PCR array was performed to determine the mRNA expressions of photocarcinogenesis-and senescence-associated genes,including p53,p21,p19,p16,pRb,fibronectin,osteonectin,smooth muscle 22 (SM22),bax,bcl-2,hypoxia-inducible factor-1 α (HIF-1α),vascular endothelial growth factor(VEGF),and human double minute-2 (hdm2).Statistical analysis was carried out by Student's t test using the software SPSS 10.0.Results The percentage of SA-β-gal-positive cells in irradiated HSF-p53 was 19.70％ ± 0.85％,significantly higher than that in unirradiated HSF-p53 (12.77％ ± 0.81％,t =6.45,P ＜ 0.05),but lower than that in irradiated control HSFs (50.48％ ± 5.30％,t =7.86,P ＜ 0.05),and similar to that in unirradiated control HSFs (18.50％ ± 0.45％,t =2.57,P ＞ 0.05).Compared with the control HSFs,the HSF-p53 showed decreased expressions of p21,p19,fibronectin,osteonectin,SM22 and bax genes (all P ＜ 0.05),but increased expressions of bcl-2,HIF-1α,VEGF and hdm2 genes (all P ＜ 0.05),and a similar expression of p16 gene (P ＞ 0.05); the repeated UVB radiation significantly promoted the expressions of p16 and pRb genes (both P ＜ 0.05),but had no obvious effect on the expressions of the other genes in HSF-p53 compared with unirradiated HSF-p53 (all P ＞ 0.05).Conclusions The inhibition of p53 expression may decelerate the UVB-induced premature senescence in HSFs,which may be involved in the p53-dependent tumor suppression.

Objective:To study the osseointegration of implant in fresh extraction socket with or without bone grafting.Methods:In the mandibular premolar region of 6 beagle dogs,bone defect in the size of (3~4)mm ×(3~4)mm ×(5 ~6)mm on the mesial wall of the mesial root socket was made.On control side implants were installed immediately into the extraction sockets(group A).On an-other side Bio-Oss grafts and membrane(GBR)were placed following implantation(group B).All animals were sacrificed 3 months af-ter implantation,specimens were examined for histo-morphometric analysis of bone to implant contact and new bone formation.Results:No implant was loosening in the 2 groups.New bone was filled in the bone defect areas in 2 groups.No statistical difference of the per-centage of new bone formation and bone-to-implant contact ration(BIC)was observed between 2 groups.Conclusion:With the defect in a certain size on the root socket wall osseointegration may occur between the new bone and implant without bone transplantation.

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.