Objective: To research the functional segments of B-FA molecule binding invariant chain and their characters. Methods:The DNA segments (α1α2, sα1α2 and α3TC ) of B-FA genes were respectively cloned and inserted into prokaryotic or eukaryotic expression plasmids,then they were singly or co-transfected with Ii gene into the engineering bacteria E. coli (BL-21)or 293T cells. After induction of expression,affinity chromatography and SDS-PAGE identification,the binding between B-FA segments and Ii molecule and co-localization in cells were observed with Pull-down and Western blot. Results:First three recombinant prokaryotic expression plasmids and four recombinant eukaryotic expression plasmids were constructed. The single molecules expressed by B-FA segments were observed after an affinity chromatography. Secondly the complexes of Ii/B-FA-α1α2 and Ii/B-FA-sα1α2 were detected by a Pull-down from the co-transfected corresponding prokaryotic expression plasmids,but no complex of Ii andα3TC,also in the western blot it was detected that B-FA-α1α2 or B-FA-sα1α2 as functional segment could bind Ii to form complex. Finally in eukaryotic expression 293T cells B-FA-sα1α2 kept localization, the same as B-FA. Conclusion: Chicken B-FA-α1α2 is function segment to bind with Ii molecule and keeps the location characters same as B-FA. The results of this research first time provide experimental evidence about B-FA functional region binding segment to Ii molecule.

The BCL6 (B-Cell Lymphoma 6) gene is a proto-oncogene that is often expressed in diffuse large B-cell lymphomas (DLBCLs). BCL6 loss of function can kill DLBCL cells, demonstrating that BCL6 is necessary for the survival of DLBCL cells and could be a therapeutic target. In this study, we found that BCL6 protein levels were consistently upregulated in DLBCL tissues, whereas its mRNA levels varied randomly in tissues, suggesting that a post-transcriptional mechanism was involved in BCL6 regulation. We used bioinformatics analysis to search for miRNAs, which potentially target BCL6, and identified specific targeting sites for miR-10a in the 3'-untranslated region (3'-UTR) of BCL6. We further identified an inverse correlation between miR-10a levels and BCL6 protein levels, but not mRNA levels, in DLBCL tumor tissue samples. By overexpressing or knocking down miR-10a in DLBCL cells, we experimentally validated that miR-10a directly recognizes the 3'-UTR of the BCL6 transcript and regulated BCL6 expression. Furthermore, we demonstrated that negatively regulating BCL6 by miR-10a suppressed the proliferation and promoted apoptosis of DLBCL cells.
3' Untranslated Regions ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Humans ; Lymphoma, Large B-Cell, Diffuse ; genetics ; metabolism ; therapy ; MicroRNAs ; genetics ; metabolism ; Proto-Oncogene Proteins c-bcl-6 ; biosynthesis ; genetics

Objective To investigate the effect of down-regulation of transient receptor potential melastatin 8 (TRPM8) on the proliferation and apoptosis of human bladder transitional cell carcinoma cells T24.Methods shRNA targeting TRPM8 was designed and synthesized, and then transfected into the T24 cells via lipofectamine 2000 mediation.The proliferation and apoptosis of T24 cells were detected with methyl thiazolyl tetrazolium (MTT) assay and flow cytometry.Expression of extracellular regulated kinase (ERK), cyclin D1, and Bcl-2 were detected with Western blot.Results TRPM8-targeted shRNA downregulated TRPM8 expression of T24 cells.MTT assay showed a significant acceleration of the proliferation of shRNA interference group compared to blank and control groups (P ＜0.01).Compared to control group, cell apoptosis rate was significantly higher in shRNA interference group (P ＜ 0.01).In addition, the expressions of PI3K, cyclin D1, and Bcl-2 were decreased in shRNA interference group.Conclusions Down-regulation of TRPM8 can induce inhibition of proliferation and promotion of cell apoptosis in human bladder transitional cell carcinoma cells T24 via regulating PI3K.It might be regarded as a novel target for clinical diagnosis and gene therapy of bladder cancer.

[ ABSTRACT] AIM:To investigate the effect of DEK downregulation on the apoptosis of gastric carcinoma SGC-7901 cells, and to explore its associations with NF-κB signaling pathway and apoptosis related proteins.METHODS:SGC-7901 cells with different treatments were divided into 3 groups including untreated group, control siRNA group and DEK siRNA group.The expression of DEK at mRNA and protein levels in the SGC-7901 cells was detected by real-time PCR and Western blot.The cell apoptosis was examined by flow cytometry.Furthermore, the activities of caspase-3 and caspase-9 in the SGC-7901 cells were investigated by Caspase-Glo?-3/9 kit.Finally, the expression of key regulatory pro-tein p65 of NF-κB signaling pathway and apoptosis-related proteins Bcl-2 and Bax in the SGC-7901 cells was investigated by Western blot.RESULTS:Compared with untreated group and control siRNA group, the expression of DEK at mRNA and protein levels was significantly downregulated in DEK siRNA group (P<0.05).In addition, the ratios of early phase apoptosis and total apoptosis in DEK siRNA group were markedly higher than those in untreated group and control siRNA group (P<0.05).Most notably, the decrease in p65 and Bcl-2 proteins, increase in Bax protein and the increases of caspase-3 and caspase-9 activities were observed in DEK siRNA group.CONCLUSION:Downregulation of DEK mediates cell apoptosis of gastric carcinoma may be tightly associated with NF-κB signaling pathway.

Objective:To study characters of the location in cells and binding activity of chicken Ii and B-L gene expressed products.Methods:The cloned gene segments of chicken Ii,B-LA and B-LB were respectively inserted into prokaryotic or eukaryotic expression plasmids,and then these recombinant plasmids were respectively alone transfected or cotransfected into engineering bacteria, Rosetta(DE3) or 293T cells.All of the recombinant bacteria were induced to express and their products then were renatured.The singly expressed products were detected to their immunogenicity with Western blot, and the co-expressed products were tested their binding with pull-down method and ( SDS-) PAGE.Results:First six of prokaryotic and eukaryotic recombinant expression plasmids were con-structed.The eukaryotic expressed products of Ii, B-LA and B-LB genes located in cellular plasma membrane.The single protein molecules were achieved from prokaryotic expressed products, which were renatured and purified with a Ni-column respectively.Secondly the prokaryotic expression Ii,B-LA and B-LB molecules could respectively induce mouse to product specific anti-bodies, which could recognize the corresponding products in eukaryotic expression.This indicated that they retained their antigenicity.Finally with pull-down from the products in co-expression in engineering bacteria and renaturation the Ii/B-LA or Ii/B-LB complexes were purified and the Ii,B-LA or B-LB monomers were dissociated from these complexes after a SDS treatment.Conclusion:The prokaryotic or eukaryotic expressed products of chicken Ii and B-L genes could retain their antigenicity,and chicken Ii could bind B-L molecules after prokaryotic coexpression and renaturation.These results provide a useful method to study the relation between Ii and MHC molecules.

Objective To analyze the clinical efficacy of psychological nursing on patients with pregnancy-induced hypertension (PIH) combined with obstructive sleep apnea hypopnea syndrome (OSAHS).Methods 96 patients with pregnancy-induced hypertension (PIH) combined with OSAHS from January 2009 to July 2012 were admitted.They were randomly divided into the experimental group and the control group with 48 cases in each group.All patients were given routine drug therapy,based on this,the control group received routine care,and the experimental group was given routine care and psychological nursing.The anxiety and depression score and the treatment effect were compared between the two groups.Results After eight-week treatment,in the experimental group,the scores of SAS,SDS declined obviously and the levels of blood pressure were significantly decreased compared with those in the control group.The difference was statistically significant.Conclusions Psychological nursing for patients with pregnancy-induced hypertension(PIH) combined with OSAHS played a positive role in nursing of these patients,it can remit their anxiety and depression emotion and also can significantly decrease the patients'blood pressure.

Objective To analyze the clinical efficacy of nursing intervention on patients who were suffering from pregnancy-induced hypertension syndrome combined with obstructive sleep apnea hypopnea syndrome(OSAHS). Methods From April 2010 to December 2011,58 patients with pregnancy-induced hypertension combined with OSAHS were selected.They were randomly divided into the experimental group and the control group with 29 cases in each group.The control group received routine care and drug treatment,based upon this,the experimental group received pertinent nursing of OSAHS.The treatment effect was compared between the two groups. Results The total effective rate of the experimental group was significantly higher than the control group. Conclusions Pertinent nursing of OSAHS for patients with pregnancy-induced hypertension combined with OSAHS played a positive role in nursing of these patients,it can significantly decrease the blood pressure of patients.

Objective To evaluate the myocardial protective effect of dexmedetomidine during non-cardiac surgery in patients with coronary heart disease.Methods Eighty ASAⅡor Ⅲ patients with coronary heat disease (NYHA Ⅱ or Ⅲ)aged 43-76 yr weighing 52-80 kg scheduled for elective upper abdominal surgery were randomly divided into 2 groups(n=40 each)：control group(group C)and dexmedetomidine group(group D).Anesthesia was induced with etomidate 0.25 mg／kg，sufentanil 0.5 μg／kg and vecuronium 0.1 mg／kg.The patients were tracheal intubated and mechanically ventilated.A loading dose of dexmedetomidine 1μg/kg was injected intravenously 10 min before induction followed by infusion at 0.4 μg·kg-1·h-1 until the end of operation in group D.While equal volume of normal saline was given in group C.BIS was maintained at 40-49.Blood samples were taken before induction and at the end of operation for determination of serum concenlrations of IL-6，TNF-α，cardiac troponin Ⅰ(cTnI)and glycogen phosphorylase BB(GP-BB).The adverse cardiovascular events were recorded during operation.Results The serum concentrations of IL-6，TNF-α，cTnI and GP-BB and incidences of tachycardia and myocardial ischemia were significantly lower，while the incidences of bradycardia highcr in group D than in group C (P<0.05).Conclusion Dexmedetomidine Can exert the myocardial protective effect during non-cardiac surgery in patients with coronary heart disease and the mechanism may be related to the inhibition of the release of pro-inflammatory cytokines.

Objective To develop an accurate,rapid,high throughout genotyping method based on oligonucleotide microarray for cytochrome P450 gene polymorphisms related to paclitaxel metabolism.Methods The mutant points of 2C8 * 3,3A4 * 18 and 3A5 * 3C from cytochrome P450 gene were regarded as targets.Based on the sequences in the GenBank,the wild-type and mutant-type probes were specially designed for each mutant point.PCR primers were located in the both sides of mutant point,and furthermore the fragments of PCR products were less than 200 bp.Each type of standard plasmids was constructed.Thus,all the olignucleotide probes were modified with 3'amino-group,and the reverse primers were labeled with fluorescein (Cy3).The probes were immobilized onto certain glass slides.The specific fragments of three genes were amplified and then hybridized with oligonucleotide microarray.The results were analyzed by using certain software.Finally this assay was applied to detect 50 clinical blood specimens.Results When PCR products from standard plasmids were hybridized with DNA microarray,the corresponding probes produced positive signals.Meanwhile,the non-specific hybridization signals did not appear.The results of clinical specimens showed that the mutant rate of CYP2C8 * 3 was 2%.The point of CYP3A4 * 18 for all the clinical specimens was wild-type and the mutant rate of CYP3A5 * 3C was 62%. Meanwhile,the results from detecting 50 clinical blood specimens using oligonucleotide microarray were the same as sequencing analysis.Conclusions Oligonucleotide microarray is a reliable and accurate genotyping assay for cytochrome P450 2C8 * 3.3A4 * 18 and 3A5 * 3C polymorphisms related to paclitaxel simultaneously.This genotyping assay is a high-throughout method for guiding personalized therapy and analyzing metabolism of paclitaxel in vivo.