Objective To study the antitumor effects of dendritic cell vaccine induced by astragalus polysaccha?rides on S180 tumor-bearing mice, and its possible mechanism. Methods Dendritic cells derived from mouse bone marrow were induced maturation by astragalus polysaccharides and loaded with S180 tumor antigen to prepare tumor vaccine. Tu?mor-bearing mice were divided into four groups and treated on day-5 and day-10 respectively. Group A was injected with NS, Group B with CTX (50 mg/kg), Group C with dendritic cells induced by astragalus polysaccharides and Group D with dendritic cells induced by tumor necrosis factor (TNF)-α. After 12 days of tumor-bearing, the animals were killed. The sub?cutaneous sarcoma, thymus and spleen were separated and weighted. The inhibitory rate, thymus index and spleen index were then calculated. ELISA assay was used to detect the levels of interleukin (IL)-4, interferon (IFN)-γin serum of tumor bearing mice. Results The tumor inhibition rate was higher in astragalus polysaccharide group and cytokine group than that of CTX group (64.25%, 64.10%vs 35.11%). The thymus index was higher in astragalus polysaccharide group and cyto?kine group than that of CTX group (1.69 ± 0.26, 1.74 ± 0.38 vs 1.45 ± 0.22). The spleen index was higher in astragalus poly?saccharide group and cytokine group than that of CTX group (5.44 ± 0.76, 5.31 ± 0.81 vs 3.54 ± 0.52). The level of IL-4 was lower in astragalus polysaccharide group and cytokine group than that of CTX group (15.66±2.57, 14.72 ± 4.84 vs 23.95 ± 6.07). The level of IFN-γwas higher in astragalus polysaccharide group and cytokine group than that of CTX group (16.54 ± 3.71, 17.20 ± 2.03 vs 10.37 ± 2.19). All the differences were statistically significant (P<0.05). Conclusion Dendritic cell vaccine induced by astragalus polysaccharides can effectively inhibit tumor growth. Its mechanism may be associated with the promotion spleen index and thymus index of S180 tumor-bearing mice, the effective correction of Th1/Th2 imbalance in?duced by tumor, and the enhancement of antitumor immune responses.

ObjectiveTo prepare high specific monoclonal antibodies(mAbs) against BP26 of Brucella(B.)melitensis.Methods A recombinant plasmid pET-28a-BP26 was constructed and transformed into competent Escherichia coli BL21 (DE3),and then the bacteria were induced by 1 mmol/L isopropylthio-β-D-galactoside (IPTG).After induction,the recombinant BP26 protein (rBP26) was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PGAE) and nickel ion affinity chromatography(Ni-NTA).Mice were inoculated with rBP26 antigens for three times at 2-week intervals.The first subcutaneous injection contained 100 μg rBP26 with 0.1 ml complete Freund adjuvant.The second subcutaneous injection was 50 μg rBP26 with 0.1 ml incomplete Freund adjuvant.The antibody titers to rBP26 were determined 2 weeks after each reimmunization.Three days before cell fusion,the mice with the highest titer were intraperitoneally injected with 50 μg rBP26 in 0.1 ml PBS.Pre- and post-immunization sera were collected and used as negative or positive controls for screening mAbs.Mice with the highest titer were sacrificed and spleen cells were isolated.The spleen cells of rBP26 immunized mice were fused with SP2/0 myeloma cells in a ratio of 5 ∶ 1 by polyethylene glycol(PEG) 1450.Antibody-producing hybridomas were primarily screened by an indirect enzyme-linked immunosorbnent assay(ELISA) with rBP26.Reactive hybridomas were subcloned for 3 times,then the strains of hybridoma cells secreting antibodies against BP26 were obtained.Supernatant of cloned hybridoma cultures was collected for mAb analyses.These mAbs were named by the hybridoma clone number and tested their reactivity to membrane proteins extracted(NMP) from B.melitensis vaccine strain(M5-90) by Western blotting and Dot-ELISA.mAbs isotyping and kappa(κ) or lambda(λ) light chain was identified by Mouse Monoclonal Antibody Isotyping Kit.Results A total of two mAbs reactive to rBP26 of B.melitensis were selected from antibody screening hybridomas by indirect-ELISA.The two mAbs were named 3C3 and 5A5,and identified as IgG1 (κ) and IgG2(κ),respectively.They could react with NMP from M5-90.Conclusions Results of identification show that two mAbs against rBP26 can be produced.The two mAbs can recognize natural BP26 protein,giving the experimental materials for further research on identification of its epitopes.

Objective To develop a liquid bandage for self aid of war wound.Methods Solution A and B were prepared containing functional polymers derived from the biocompatible poly (ethylene glycol) and hyaluronic acid,which had ability of fast crosslinking reaction when they were mixed.Among them,A solution mainly comprised of mercapto group-containing hyaluronic acid derivative (HA-SH),water-solubility starch,propylene glycol,benzoic acid and etc.B solution contained pentaerythritol tetraacrylate derivative (4arm-PEG-Acrylate) and benzoic acid.The structure of mercapto group-containing hyaluronic acid derivative was confirmed by IR,1H-NMR and etc.HA-SH and 4arm-PEG-Acrylate were formulated to different concentrations (W/V) in different buffers.The two kinds of solution were mixed in different ratios,and in situ crosslinking hyaluronic acid hydrogel was obtained;the crosslinking time was recorded.The adhesive force and waterholding capacity of the hydrogel after crosslinking were measured by adding excipients such as water-solubility starch.Results In case the concentration of HA-SH and 4arm-PEG-Acrylate was 2％ and pH value was 11,the hydrogel film on wound skin would be quickly formed in 20 seconds for wound dressing after spraying A and B solution onto the wound skin.Conclusion The liquid bandage has strong adhesive force,high water-holding capacity and controllable crosslinking time,so it could be used as a novel wound dressing for war wound.

A temperature control system for quantitive polymerase chain reaction (PCR) is presented in the paper with both software and hardware configuration. The performance of the control system has been improved by optimizing the software and hardware design according to the system's properties. The control system has been proven to have a good repeatability and reliability as well as high control precision.
Equipment Design ; Microcomputers ; Polymerase Chain Reaction ; instrumentation ; Software ; Software Design ; Temperature

10.3969/j.issn.2095-4344.2013.25.004

Fatty acids of Ralstonia solanacearum cultured under different temperatures, times, pH values and cultural media were detected by using gas chromatography (GC) method. Rs-J.1.4-010704-01v, a viru- lent strain of R. solanacearum isolated from ginger was chosen for the experiment. The results showed that the kind of fatty acid of Rs-J.1.4-010704-01v fluctuated from 14 to 33. The contents of its three plentiful fatty acids, C16:1?7c/C15:0 ISO 2OH, C16:0 and C18:1?7c (with retain times of 10.644, 10.950 and 14.177 min, respectively), also varied in a range of 55.66% to 75.69%. The diversification of the bacterium’s fatty acids at various cultural conditions was clustered into four groups by cluster analysis, according to the kinds and percentage contents of the fatty acids detected. The pathogenicities of Rs-J.1.4-010704-01v under 20?C and 25?C were deduced to be mid-virulent, with C16:0 less than C16:1?7c/C15:0 ISO 2OH. The bac- terium showed as a virulent strain under the other cultural conditions including 30?C~40?C, 24 h~96 h, pH 5~9 and four cultural media (LB、NA、TTC and TSB), with C16:0 more than C16:1?7c/C15:0 ISO 2OH.However, the difference between C16:0 and C16:1?7c/C15:0 ISO 2OH raised significantly from 2.35 to 13.23 under 40?C and 48 h~96 h. Meanwhile, the kind of fatty acid increased more than 30 as the cultural time increased. It was concluded that temperature and cultural time had more significant effects on the fatty acid composition of R. solanacearum than pH value and cultural medium.

OBJECTIVETo explore the expressions of HO-2 and CO in the corpus cavernosum of castrated rats in order to further study the pathogenesis of erectile dysfunction (ED).

METHODSWe randomly divided 72 male SD rats into four groups: normal control, sham operation, castration, and castration + ZnPP. We detected intracavernous pressure (ICP) and penile erection in the basic condition and after apomorphine (APO) induction, determined the expression of the HO-2 protein in the corpus cavernosum by laser scanning confocal microscopy, and measured the level of CO by spectrophotometry during different periods of penile erection.

RESULTSThe ICP in the basic condition and that after APO induction and the rate of penile erection were decreased significantly in the castration group ([11.68 ± 0.69] mmHg, [54.81 ± 3.86] mmHg, and 33.3%) and the castration + ZnPP group ([11.20 ± 0.71] mmHg, [41.17 ± 5.41] mmHg, and 22.2%) as compared with the normal control ([22.83 ± 2.66] mmHg, [66.92 ± 7.77] mm-Hg, and 100%) and the sham operation group ([23.35 ±2.22] mmHg, [70.43 ?7. 22] mmHg, and 100%) (all P <0. 01). After APO induction, ICP in the castration + ZnPP group was remarkably reduced in comparison with that in the castration group (P < 0.01), and so was the expression of the HO-2 protein before and during penile erection in the castration (445.4 ± 23.7 and 847.4 ± 35.0) and the castration + ZnPP group (390.1 ± 29.7 and 526.0 ± 52.5) compared with the normal control (512.7 ±57.4 and 1145.2 ± 89.8) and the sham operation group (583.7 ± 8.0 and 1016.3 ± 79.8), the expression of the HO-2 protein significantly decreased in the castration group (445.4 ± 23.7 and 847.4 ± 35.0) (P < 0.05 or 0.01), markedly lower in the castration + ZnPP than in the castration group during penile erection (P < 0.01) but with no significant differences among the four groups after it. Before, during and after penile erection, the levels of CO were remarkably decreased in the castration ([20.59 ± 1.01], [32.53 ± 1.26], and [18.71 ± 1.22] x 10(-7) nmol/L) and the castration +ZnPP group ([12.52 ± 1.05], [21.90 ± 1.02], and [16.56 ± 0.55] x 10(-7) nmol/L) as compared with the normal control ([26.76 ± 1.41], [48.25 ± 1.01], and [27.10 ± 1.58 ] x 10(-7) nmol/L) and the sham operation group ([25.41 ± 2.09], [ 47.90 ± 1.22], and [25.67 ± 1.20] x 10(-7) nmol/L) (P < 0.05 or 0.01), significantly lower in the castration + ZnPP than in the castration group during penile erection (P < 0.01).

CONCLUSIONDecreased expressions of HO-2 and CO may correlate with erectile dysfunction in castrated rats.

Animals ; Apomorphine ; pharmacology ; Carbon Monoxide ; metabolism ; Dopamine Agonists ; pharmacology ; Erectile Dysfunction ; etiology ; Humans ; Male ; Molecular Chaperones ; metabolism ; Orchiectomy ; Penile Erection ; drug effects ; Penis ; drug effects ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Objective To observe the effects of 75 gram glucose oral tolerance test (75 g OGTT) and standard mixed meal test (SMMT) on insulin secretion function of the islets of Langerhans and plasma free fatty acid (FFA) in patients with type 2 diabetes mellitus.Methods Seventy-six patients with type 2 diabetes without using insulin and with no obvious complications were recruited for 75 g OGTT following overnight fasting on the first day and SMMT (bread 50 g,egg 50 g and milk 250 ml) on the 7th day.Blood specimens were collected from each patients before the tests and 30 min,60 min,120 min and 180 min after glucose or meal load to measure their levels of plasma glucose,serum insulin,C peptide,FFA and lipids (total cholesterol,triglyceride,high-density and low-density lipoprotein cholesterol).Results No difference in fasting plasma glucose,serum insulin,C peptide,FFA and lipids between 75 g OGTT and SMMT was found.Postprandial plasma glucose 30 min,60 min,120 min and 180 min after 75 g OGTT was significantly higher than that after SMMT,with (15.3?3.5) vs (9.9?3.4) mmol/L,(18.2?4.8) vs (12.8?4.0) mmol/L,(16.3?5.8) vs (12.2?4.9) mmol/L and (10.6?5.4) vs (9.5?4.5) mmol/L (F=28.1,P

OBJECTIVETo observe the effect of Huatuo Zaizao extractum (HTZZ) on focal cerebral ischemia/reperfusion (I/R) neurogenesis in rats induced by middle cerebral artery occlusion (MCAO) and its mechanism.

METHODTotally 55 healthy adult male Sprague-Dawley rats were divided into the sham operation group, the MCAO model group and HTZZ high, middle and low dose groups (5, 2.5, 1.25 g x kg(-1)), with 11 rats in each group, and orally administered with drugs. The focal cerebral ischemia model was established by performing a middle cerebral artery occlusion (MCAO, 90 min) followed by a seven-day reperfusion (once a day). The neurogenesis and expressions of extracellular signal-regulated kinase (ERK) and cAMP response element binding protein (CREB) were detected by the immunofluorescent staining. The enzyme linked immunosorbent assay (ELISA) was adopted to determine the vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF).

RESULTMCAO (90 min) followed by a seven-day reperfusion resulted in the significant increase in the number of penumbra cortex newborn neurons (BrdU(+) -NeuN(+)), which was accompanied by the growth of ERK and CREB phosphorylation and VEGF and BDNF levels. HTZZ could promote the generation of newborn neurons (BrdU(+)-NeuN(+)) and the ERK and CREB phosphorylation and increase VEGF and BDNF levels at the ischemic side.

CONCLUSIONHTZZ could promote the neurogenesis, which may be the interventional targets of effective traditional Chinese medicine Huatuo Zaizao extractum in promoting the self-repair function of the cerebral ischemic areas.

Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; physiopathology ; Brain-Derived Neurotrophic Factor ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Male ; Neurogenesis ; drug effects ; Neurons ; cytology ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

OBJECTIVETo explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS).

METHODSPeripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay.

RESULTSThe morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio.

CONCLUSIONAPS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

Antigen-Presenting Cells ; cytology ; drug effects ; immunology ; Cancer Vaccines ; immunology ; Cell Line ; Cell Proliferation ; drug effects ; Coculture Techniques ; Dendritic Cells ; cytology ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-12 ; immunology ; Leukocytes, Mononuclear ; cytology ; immunology ; Lymphocyte Activation ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects