Objective:To compare the human ??T lymphocytes expanded with HSP 70BCG and HSP 70 m-peptide complexes with those expanded with solid phase antibody in vitro.Methods:PBMCs were cultured with anti-TCR ?? antibody?HSP 70BCG and HSP 70 m -peptide complexes.The total cell number was counted.The flow cytometer was used to analyze the lymphocytes phenotypes and subtypes.RT-PCR was used to analyze the level of V? mRNA.The cytotoxitic activity of ??T cells against Daudi was determined using MTT calorimetric assay.Results:After 14 days of culture,in the antibody culture system,the total cell number increased about 30-40 fold,and the ratio of ??T cell reached to 86.3%;In the HSP 70BCG culture system,the total cell number increased about 7-8 fold,and the ratio of ??T cell reached to 71.23%;In the HSP 70-peptide complexes culture system,the total cell number increased about 4-5 fold,and the ratio of ??T cells reached to 27.26%.The antibody and HSP 70BCG activated ??T cells possessed a whole repertoire and mainly expressed V?9/V?2 subset and exhibited a strong cytotoxicity against Daudi.Conclusion:Anti-TCR ?? antibody and HSP 70BCG were able to proliferate purer ??T cells.The cells had a whole repertoire,and exhibited strong cytotoxicity against Daudi.

Objective To investigate the effect of cimetidine on the clonal expansion of TCR V? subfamily of T cells in cord blood after the stimulation by K562 cells in vitro.Methods Cimetidine(1?10-5 mol/L) or K562 cells(1?106/ml) or both of them were respectively cultured with mononuclear cells(MNC) isolated from normal human cord blood for 2 weeks.After the induction,specific cytotoxicity of the proliferated T cells were detected with K562 cells as the target cells.The selective usages and clonal expansion of TCR V? subfa-mily of T cells were analyzed by RT-PCR and genescan technique.Results After induction for 2 weeks,the 3 groups showed the increased cell proliferation,in which specific cytotoxicity of T cells induced by both cimetidine and K562 cells against K562 cells was enhanced significantly compared with the other 2 groups(P

Objective To systematically review the effectiveness and safety of pantoprazole ( PAN ) vs. ranitidine (RAN) for patients with gastroesophageal reflux disease (GERD). Methods PubMed,Medline,EMbase,The Cochrane Library and three Chinese literature databases (CNKI,VIP and Wan fang) were retrieveed.Randomized controlled trials (RCTs) which compared the clinical outcomes of PAN group vs. RAN group for GERD were included. Two reviewers independently screened literatures in accordance with the inclusion and exclusion criteria, extracted the data and assessed the methodological quality of included studies.Then,meta-analysis was performed using RevMan 5.2 software. Results A total of 8 RCTs involving 1 590 patients were included.The results of meta-analysis showed that the PAN group was significantly superior to RAN group in terms of the healing rates and the relief rates of chief symptom for GERD of gradeⅠ-Ⅲ. While there was no significant difference in the incidence of adverse events between the two groups [GradeⅠ,RR=1.17,95%CI (0.80,1.70),P=0.43;GradeⅡorⅢ, RR=0.76,95%CI (0.43,1.36);P=0.36]. Conclusion Current evidence indicates that,pantoprazole is more effective than ranitidine for GERD of grade Ⅰ-Ⅲ,but both treatments are safe and well tolerated.

New strategies to improve vaccine efficacy against human immunodeficiency virus type 1 (HIV-1) are still required. DNA vaccines, exhibiting potential advantages over conventional vaccines for their simplicity and versatility, can induce specific humoral and cellular immune responses. We developed a recombinant pVAX1 DNA vector carrying p24 gene of HIV-1. The results showed that pVAX1 mediated gene possessed the ability of effective expression in both transfected 293T cells and BALB/c mice. And pVAX1-p24 DNA prime and boost immunization can induce significant P24-specific humoral immune responses and cellular immune responses in BALB/c mice. Furthermore, immunization with pVAX1-p24 DNA prime and protein boost induced 7.3 to 8.0-fold greater p24-specific humoral responses than pVAX1-p24 DNA prime and boost, while the cellular immune responses induced by combined immunization was lower. The results suggested that pVAX1-p24 DNA and P24 protein vaccine is a promising HIV-1 vaccine, and the selections of the immunization strategies are important for the immunization results.
AIDS Vaccines ; genetics ; immunology ; Animals ; DNA ; genetics ; immunology ; HEK293 Cells ; HIV Core Protein p24 ; genetics ; immunology ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; genetics ; immunology

Radiotherapy is a treatment for cancer with undesired by-effects. In order to develop a new radiation protective agent that could reduce the by-effects, we tried to express and purify human cryptochrome 1 (hCRY1). The coding sequence of hCRY1 was inserted into prokaryotic expression plasmid pET28a(+), and this protein was purified from Escherichia coli BL21(DE3) after IPTG induction, ultrasonication, inclusion body dissolution, gradient dialysis, nickel column purification and ultrafiltration. The yield of hCRY1 in 1 L E. coli culture (LB medium) was about 10-15 mg. The radiation protective efficiency of hCRY1 was monitored by detecting X-ray-induced H2A.X foci in HaCaT cells. The results of immunofluorescence show that hCRY1 significantly reduces X-ray stimulated DNA damage response. The apoptosis of HaCaT cell was also detected, and the repression of H2A.X foci formation was not due to hCRY1's cytotoxity. All these data suggest a potential application of recombinant hCRY1 as a protective agent for radiotherapy.
Cryptochromes ; biosynthesis ; Escherichia coli ; Humans ; Plasmids ; Radiation-Protective Agents ; Recombinant Proteins ; biosynthesis

Polyethylenimine (PEI) is one of the most characterized non-viral vectors. It can condense DNA in a good manner and achieve high transfection efficiency. Minicircle DNA (mc-DNA) is a novel kind of supercoiled DNA which is devoid of bacterial backbone. mc-DNA is superior to conventional DNA for its higher transfection efficiency and longer time-span. In this study, we combined PEI and mc-DNA in gene delivery system. We investigated the physicochemical and biochemical effects of this non-viral system and further explore its potential in tumor gene therapy. mc-DNA was obtained by recombination of parental plasmid in the presence of L-arabinose, and complexed with PEI. The results of transmission electron microscopy and scanning electron microscopy showed that the particles were spherical and homogeneous. Through gel retardation assay and MTT assay, we found that there were no obvious differences in binding capability of PEI to mc-DNA and plasmid DNA, as well as in cytotoxicity. The results of dynamic light scattering showed that the size of PEI/mc-DNA was about 68 nm, a slight larger than that of PEI/plasmid DNA. Furthermore, the tumor cells transfected with mc-GFP showed higher GFP expression level than that of conventional plasmid. The same results were achieved in the cells treated with tumor-suppressor gene pten, assayed by RT-PCR and Western blot. It indicates that the system of PEI/minicircle DNA is promising in gene transfer.
DNA, Circular ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Nanoparticles ; chemistry ; PTEN Phosphohydrolase ; genetics ; Polyethyleneimine ; metabolism ; Transfection

Objective To evaluate the clinical efficacy of Wuling San in the treatment of Chronic heart failure by meta analysis. Methods The randomized controlled trials were searched from CNKI, Wanfang Data, VIP Data, CBM Data, Cochrance Library, PubMed, EMbase. Review 5.2 Manager software, stata 14.0 software, GRADE profiler 3.6.1 software were used to bias risk assessment, meta analysis, and quality evaluation of the studies. Results Fifteen studies with 1160 patients were included. The overall quality of studies is low. The meta analysis results showed that the total efficiency of improving heart function in the treatment group were superior to the control group [OR=4.60, 95％ CI (3.12, 6.77), Z=7.72, P<0.01]. Due to the heterogeneity, the studies of left ventricular ejection fraction (LVEF) were divided into two subgroups according to the course of treatment, one group with more 4 weeks course treatment and the other group with less 4 weeks. The LVEF in the treatment group was superior to the control group [WMD=9.53,95％ CI (8.80, 10.28), Z=25.63, P<0.01]. The brain natriuretic peptide (BNP) iin the treatment group was superior to the control group [WMD=-65.52, 95％ CI (-88.45, -48.59),Z=6.74,P<0.01]. Conclusions The present meta-analysis suggests that combination of conventional western medicine and Wuling San has better effect on heart failure. Due to the poor quality of included RCTs, the conclusions may exist bias. Thus, more RCTs with high quality are needed.

RNA silencing is a conserved eukaryotic pathway involved in the suppression of gene expression via sequence-specific interactions that are mediated by 21-23 nt RNA molecules. During infection, RNAi can act as an innate immune system to defend against viruses. As a counter-defensive strategy, silencing suppressors are encoded by viruses to inhibit various stages of the silencing process. These suppressors are diverse in sequence and structure and act via different mechanisms. In this review, we discuss whether RNAi is a defensive strategy in mammalian host cells and whether silencing suppressors can be encoded by mammalian viruses. We also review the modes of action proposed for some silencing suppressors.
Animals ; Gene Expression Regulation, Viral ; Gene Silencing ; Host-Pathogen Interactions ; Humans ; Mammals ; virology ; MicroRNAs ; genetics ; metabolism ; Plant Viruses ; physiology ; Plants ; virology ; RNA, Small Interfering ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Viruses ; growth & development

Porcine interferon-alpha (pIFN-alpha) fermentative production by recombinant Pichia pastoris was carried out in a 10-L bioreactor to study its metabolism changes and effects on fermentation under different inducing strategies, by analyzing the change patterns of the corresponding metabolism and energy regeneration. The results show that the specific activities of alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH) largely increased when reducing temperature from 30 degrees C to 20 degrees C under pure methanol induction, leading significant enhancements in methanol metabolism, formaldehyde dissimilatory energy metabolism and pIFN-alpha antiviral activity. The highest pIFN-alpha antiviral activity reached 1.4 x 10(6) IU/mL, which was about 10-folds of that obtained under 30 degrees C induction. Using methanol/sorbitol co-feeding strategy at 30 degrees C, the major energy metabolism energizing pIFN-alpha synthesis shifted from formaldehyde dissimilatory energy metabolism pathway to TCA cycle, formaldehyde dissimilatory pathway was weakened and accumulation of toxic intermediate metabolite-formaldehyde was relieved, and methanol flux distribution towards to pIFN-alpha synthesis was enhanced. Under this condition, the highest pIFN-alpha antiviral activity reached 1.8 x 10(7) IU/mL which was about 100-folds of that obtained under pure methanol induction at 30 degrees C. More important, enhanced pIFN-alpha production with methanol/sorbitol co-feeding strategy could be implemented under mild conditions, which greatly reduced the fermentation costs and improved the entire fermentation performance.
Animals ; Energy Metabolism ; Fermentation ; Interferon-alpha ; biosynthesis ; genetics ; Methanol ; pharmacology ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; Sorbitol ; pharmacology ; Swine

Objective To investigate the effect of up-regulation of miR-132 on expressions of angiopoietin-1 (Ang-1)/endothelium-specific tyrosine kinase receptor 2 (Tie2) in focal cerebral ischemia reperfusion injury of rats.Methods Forty adult healthy SD rats were randomly divided into sham-operated group,cerebral ischemia group,miR-132 mimic group and negative control group (n=10).The models of middle cerebral artery occlusion in the later three groups were established by using modified Longa suture method.Rats in the miR-132 mimic group and negative control group were injected miR-132 mimic 15 μg and negative control 15 μg via paracele.Rats in each group were sacrificed 24 h after ischemia,and the brain tissues were collected;the total infarct volumes were calculated by TTC staining.The mRNA expressions ofmiR-132,Ang-1 and Tie2 in ischemic cerebral cortex tissues were detected by real-time fluorescence quantitative PCR.The protein expressions of Ang-1,Tie2,CD31 and vessel endothelial growth factor (VEGF) in ischemic cerebral cortex tissues were detected by Western blotting.Results The total infarct volume in the miR-132 mimic group was (27.92±3.05) mm3,which was significantly smaller than that in the cerebral ischemia group and negative control group ([51.34±2.86] mm3 and [50.46±2.57] mm3,P＜0.05).The relative mRNA expression levels of miR-132,A ng-1,Tie2,and the protein expression levels of Ang-1,Tie2,CD31,VEGF in the ischemic cerebral cortex tissues of the miR-132 mimic group were significantly higher than those in the negative control group,cerebral ischemia group and sham-operated group (P＜0.05);and those in the negative control group and cerebral ischemia group were significantly higher than those in the sham-operated group (P＜0.05).Conclusion Up-regulation ofmiR-132 expression could improve the ischemic states of ischemic stroke in rats,which might be related to Ang-1/Tie2 increased expressions to promote angiogenesis in ischemic brain tissues.