Objective To evaluate the utility of fluorescent dye SYTO 13 for high -resolution melting ( HRM) detection in single nucleotide polymorphism ( SNP) genotyping and its clinical application . Methods This is a performance verification study .36 genotype defined samples were divided into three groups:SNP rs3125734 C>T (class Ⅰ SNP) ,rs255758 A>C (class ⅡSNP) and rs688C>T.These samples were used to evaluate SYTO 13′s SNP genotyping capability of class ⅠSNP, classⅡSNP, and two PCR products of different lengths (52 and 107 bp) covering the same SNP of rs688C>T.The commercial HRM dye of LCGreen Plus was used as the control .The genotyping capability is indicated by the Tm difference(ΔTm) between wild type and homozygous mutant genotypes .The Tm differences between wild genotype and homozygous mutant genotype were compared using the Independent Samples t test.Paired t test was used to evaluate genotyping capability of the two dyes .The clinical applicability is evaluated by synchronously performing PCR amplification and HRM analysis on thirty -five randomly selected DNA samples with known genotypes of the three SNPs .Results The SNPs of class Ⅰ and class Ⅱ can be genotyped directly and clearly with SYTO13 (ΔTmclas Ⅰ =0.36 ±0.05,tclas Ⅰ =14.827,Pclas Ⅰ =0.000;ΔTm clas Ⅱ =0.42 ±0.110,tclasⅡ =9.539,Pclas Ⅱ =0.000).The classⅠSNP genotyping results was better using SYTO13 (ΔTmSYTO13 =0.39 ±0.027), while the SNP genotyping for small amplicon did not discriminated clearly in this study .Long amplicons of class ⅠandⅡSNPs can be identified directly except for several samples which can be genotyped accurately after having performed reexamination .Conclusion SYTO13 can apply for HRM analysis of genotyping classⅠand ⅡSNPs with long amplicon and for clinical routine detection.

Objective To investigate the nerve recanalization and the motor function of hind legs after transplantation of peripheral nerve grafts treated with microsurgical technique at chronic spinal cord injury (SCI) in rats.Methods The SD rats were established into SCI model with improved Allen method.The rats were divided into two parts 12 weeks after the injury.In experimental group:by microsurgieal technique. the sural nerves were removed epineufium and transplanted into SCI lesion,control group rats were treated without any operation.Retrograde HRP tracing through sciatic nerve were practiced at 1 month,2 month,3 month after transplantation of peripheral nerve grafts.The morphological changes were observed at section of spinal cord and the motor functions of both hind legs of rat were detected.Results The morphology of the injured spinal cord sections turned better.Retrograde HRP tracing through sciatic nerve showed some HRP positive markers at the site of near rostral end of the nearly injured part at one month after transplantation and increased with the time going by.Motor function of hind legs of rats recovered significantly in transplantation groups.Conclusion Peripheral nerve grafts treated with mierosurgical technique have repairing effect on chronic spinal cord injury in rats.

Objective To explore the clinical value of renal dynamic imaging and urinary N-acetyl-?-D-glucosaminidase(NAG),apoptosis DNA fragment(ADF) in evaluating the damage degree of hydronephrotic kidneys(HnK) in children with hydronephrosis.Methods Level of glomerular filtration rate(GFR) was detected in 41 children with congenital hydronephrosis by renal dynamic imaging,and urine NAG,ADF in pelvis in HnK and healthy kidneys (as controls) were detected by enzyme-linked immuno-sorbent assay(ELISA).Patholo-gic changes of HnK in 41 children were graded intoⅠ~Ⅴ according to Elder standard. And GFR,urinary NAG and ADF of HnK were divi-ded into subgroup according to pathologic changes ,at the same time statistical analysis was performed within each groups. And the correlations of pathologic grades with GFR,urinary NAG and ADF of HnK were analyzed.Results 1.Kindneys GFR in healthy kidneys and Hnk were (174.33?20.43)?10-3 L/min,(143.86?17.51)?10-3 L/min respectinely,and there was significant difference between healthy kidneys and Hnk (P0.05).3.There was significant negative correlation between GFR levels of HnK and pathologic grades(r=-0.814 P0.05).Conclusions For hydronephrotic kidneys,urinary NAG can eva-luate impaired nephric tubule whereas renal dynamic imaging may evaluate the damage level of glomeruli;urine ADF may not indicate the damage level of diseased kidneys in children with congenital hydronephrosis.

Behcet Syndrome ; complications ; Hemorrhage ; etiology ; Humans ; Pharynx

OBJECTIVETo discuss the anti-tumor effect of Xihuang pill on tumor-bearing rats and its effect on the immune clearance function of tumor-bearing organisms.

METHODWalker256 tumor cells were adopted to establish the tumor-bearing rat model. The rats were randomly divided into five groups: the normal control group, the model control group, the lentinan group and Xihuang pill low dose, middle dose and high dose groups, with 10 rats in each group, and continuously treated and given drugs for 14 d after modeling. Blood and tumors were collected from abdominal aorta to calculate the tumor inhibition rate. The content of CD3+, CD4+, CD8+ T cells and adhesion molecule B7-1 (CD80) in peripheral blood were detected by flow cytometry (FCM). The expressions of IL-2 and IFN-gamma in were determined by ELISA.

RESULTThe tumor inhibition rate of the Xihuang pill high dose group was 33. 1 percent. Compared with the model group, the Xihuang pill large dose group showed significantly low IL-2, IFN-gamma, CD3+, CD4+, B7-1 in peripheral blood, with statistical significance in their differences (P < 0.05).

CONCLUSIONXihuang pill could show its anti-tumor effect by enhancing the immune clearance function and increasing IL-2, IFN-gamma, CD3+ T, CD4+ T, B7-1 in peripheral blood.

Animals ; Antineoplastic Agents ; administration & dosage ; B7-1 Antigen ; genetics ; immunology ; Breast Neoplasms ; drug therapy ; genetics ; immunology ; CD4-Positive T-Lymphocytes ; drug effects ; immunology ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Flow Cytometry ; Humans ; Immune System ; drug effects ; Immunologic Factors ; administration & dosage ; Interferon-gamma ; genetics ; immunology ; Interleukin-2 ; genetics ; immunology ; Rats ; Rats, Wistar ; Tumor Burden ; drug effects

OBJECTIVETo analyze the drift of T cell receptor (TCR) Vα and Vβ gene family CDR3 spectratype in response to ovarian carcinoma cells mediated by an anti-human ovarian carcinoma/CD3 bispecific single-chain antibody (BHL-1), and explore the mechanism of the bispecific single-chain antibody-mediated T cell immune response.

METHODSImmunoscopic spectratyping technique was used to analyze the TCR repertoire diversity (CDR3 spectratype distribution) of the T cells from 6 healthy donors before and after stimulation of the cells with human ovarian carcinoma in the presence of BHL-1. The predominant usage of TCR α and Vβ chain CDR3 was analyzed after the stimulation, and sequence analysis was performed for the CDR3 region of the monoclonal T cells.

RESULTSThe spectratypes of Vα and Vβ gene family TCR CDR3 region showed a Gaussian distribution before stimulation of the T cells from the 6 donors. After stimulation of the T cells, CDR3 spectratype drift occurred in the T cells, and some TCR Vα and Vβ families showed an anomalous and oligoclonal expansion. Different CDR3 sequences of the Vα and Vβ gene family TCR were found in the monoclonal T cells stimulated with BHL-1.

CONCLUSIONCDR3 spectratype drift occurs in TCR α and Vβ chains of T cells after stimulation with human ovarian carcinoma cells and BHL-1, indicating that the predominant usage of TCR Vα and Vβ families is associated with the specific T cell immune response mediated by BHL-1.

Antibodies, Bispecific ; immunology ; Cell Line ; Cell Line, Tumor ; Complementarity Determining Regions ; immunology ; Female ; Humans ; Monocytes ; immunology ; Ovarian Neoplasms ; immunology ; Receptors, Antigen, T-Cell, alpha-beta ; immunology ; Single-Chain Antibodies ; immunology

Objective To investigate clinical manifestation of unspecified functional bowel disorder (UFBD), the features of coexistence with functional gastrointestinal disorder (FGID) and its relationship with psychological factors and sleep disturbance in the Chinese Army servicemen.Methodsc FGIDs were diagnosed based on the RomeⅢ Modular Questionnaire. The subjects were 189 servicemen with UFBD (UFBD group) and 372 without FGID (control group). All subjects completed symptom checklist 90 (SCL-90) and Pittsburgh Sleep Quality Index (PSQI) questionnaire.Results'Have to rush to the toilet when having a desire to defecate' was the most frequent symptom of UFBD (93.7%). More than one half of UFBD patients had the symptom 'a feeling of incomplete emptying as bowel movements' or 'straining during bowel movements'. Twenty-eight percent of UFBD subjects had combined FGID (namely cFGID). Among them, the most frequent was proctalgia fugax (7.9%), followed by cyclic vomiting syndrome (6.3%), functional fecal incontinence (6.3%), functional dyspepsia (4.8%) and belching (4.8%). The UFBD group scored significantly higher than the control group in the global severity index (GSI) and in all SCL-90 subscales (P<0.05). The scores of the four domains (sleep quality, sleep latency , sleep disturbance and daytime function disorder), total PSQI score and proportion of poor sleeping quality were significantly higher in the UFBD group than in the control group (P<0.05). The subjects scored significantly higher in combined FGID group than in UFBD group in GSI and in all of SCL-90 subscales, except for phobic anxiety subscales (P<0.05). However, there was no significant difference in each domain, total PSQI and proportion of poor sleeping quality between the cFGID group and UFBD group (P>0.05).ConclusionPathogenesis of UFBD may be closely correlated with psychiatric and psychological factors and sleep disturbance. cFGID are associated with an increased severity of psychopathological features.

Toad venom is the Bufo bufo gargarizans or B. melanostictus after the ears of the gland secretion, used in the treatment of various cancers in recent years. Research shows that the main anti-tumor components in bufadienolide. Bufadienolide have free type structure and conjunct type structure. To identify and clarify the difference between bufogenin and bufotoxin contained in Bufonis Venenum, which was from B. bufo gargarizans, an UPLC-TQ-MS method has been established. UPLC-TQ-MS method was used to identify and quantify the major bufadienolides in Bufonis Venenum. UPLC-TQ-MS assay with positive ion mode was performed on a Waters ACQUITY UPLC BEH C, (2.1 mm x 100 mm, 1.7 µm) with the mobile phase consisting of 0. 1% aqueous formic and acidacetonitrile in gradient elution at a flow rate of 0.4 mL · min⁻¹ and the column temperature was set at 35 °C. By comparing their retention time and high resolution mass data of Bufonis Venenum extracts, 37 effective components were primarily identified by MS/MS analysis in positive ion mode. Twenty-six of them were free-type bufadienolides (bufogenin), 11 of them were conjugated bufadienolides. There were significant differences in the main composition between fresh and processed Bufonis Venenum. The study found that the chemical composition of toad venom through great changes after processing, conjunct type content is much less, free type content as well change.
Amphibian Venoms ; chemistry ; metabolism ; Animals ; Bufonidae ; classification ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Molecular Structure ; Tandem Mass Spectrometry ; methods

Objective　To explore the effect of video-assi sted thoracic surgery (VATS) on common diseases of chest. Methods　Video-assisted thoracoscopic surgery was performed on 131 patients with ches t diseases from April 1994 to December 2000 in which 109 cases were spontane ous pneumothorax and hemothorax, 10 pulmonary tuberculoma, 5 pulmonary carcinoma , 3 esophageal carcinoma, 2 localized benign mesothelioma, 1 pulmonary hamartoma and 1 myasthenia gravis. Results　There was no operative death in all cases. Four patients with spontaneous pneumothorax complicated persist ent air leak(more than 7 d) and 1 patient with hemopneumothorax formed hemoth orax after the operation, which was stopped by the second VATS. The others recov ered well without any postoperative complications. Conclusion　 VATS is characterized by safety and mild tissue injury in the operation, and les s pain, fewer complications, rapid recovery and short duration of hospitalizatio n after the operation.

Objective:To comparison of three different beta blockers on murine hemangioma (EOMA cells) cells in vitro and in vivo effects.Preliminary study on the therapeutic effect of propranolol on vascular tumor in mice and possible mechanisms , provide a reference for beta blockers in the treatment of infantile hemangioma .Methods: Comparative study on the effects of three kinds of different β-receptor blockers---metoprolol, propranolol and butoxamine , on the proliferation and apoptosis of Mouse Hemangioendothelioma Endothelial cell (EOMA cells) was conducted in vitro.EOMA cells were cultured in vitro,randomly divided into different groups,propranolol and timolol were added into the medium respectively ,after 24 h intervention.MTT assay and acridine orange staining assay were conducted respectively to detect cell viability and apoptosis level .EOMA cells were transplanted into nude mice in vivo.Tumor volume growth to 100 mm3 ,animals were randomly divided into 4 groups respectively ,the control group ,metoprolol group,Bhutto Samin group and propranolol group ,drug group according to 2 mg/( kg? d) oral gavage ,control group were given an equal volume of saline ( NS ) , every two days measurement tumor volume size .Serum levels of tumor necrosis factor alpha ( TNF-α) and vascular endothelial growth factor ( VEGF ) were detected by enzyme linked immunosorbent assay ( ELISA ) in the end of the experiment.Results:For propranolol,after 24 h treatment,significant differences of cell viability and apoptosis were noted (P<0.05) at the concentration of 50 μmol/L,while continuing to increase to 800 μmol/L,the cell survival rate decreased sharply to close to 10%. Acridine orange staining at the 50 μmol/L group after 24 h revealed many apoptotic cells .For metoprolol and butoxa mine ,significant differences of cell viability and apoptosis were noted ( P<0.05 ) at the concentration of 100 μmol/L,while continuing to increase to 800μmol/L,the cell survival rate decreased sharply to close to 20%.It was significantly higher than propranolol group at the same concentration ( P<0.05 ) .It showed a similar trend in acridine orange staining .In vivo experiments showed that the end of the experiment of metoprolol , butoxamine group and propranolol drugs in mice tumor volume , respectively ( 1 642.8 ±89.3 ) , ( 1 529.3 ± 119.1) and (752.7±46.5)mm3,significantly lower than the control group of mice tumor volume of (2 023.3±123.0) mm3(P<0.001).Metoprolol,butoxamine mice and propranolol drugs group ,serum VEGF levels for (606.5±105.8 ) pg/ml,(534.3±243.2 ) pg/ml and (420.1±123.7) pg/ml, significantly lower than the PBS control group [(825.8±145.7) pg/ml,(P<0.05)],the TNF alpha result was followed by(301.3±62.3) pg/ml,(305.1±53.8) pg/ml and (288.8±59.5) pg/ml,significantly lower than the normal control group [(444±100.4) pg/ml,P<0.05].Conclusion:Three kinds of beta-blockers can effectively inhibit EOMA cells proliferation and induce apoptosis in vitro, the role of propranolol more significantly than butoxamine and metoprolol .Three kinds of beta blockers restrain the growth of the hemangioma in vivo ,in which the inhibitory effect of propranolol is stronger than the metoprolol and butoxa mine.Three kinds of beta blockers can lower the levels of VEGF and TNF-αin vivo.Indicating that propranolol on vascular tumor in mice may be one of the mechanisms of β1 and β2 receptor synergy effect and its mechanism in the treatment of hemangioma may be associated with VEGF and TNF-α.