OBJECTIVETo analyse the WT1 expression and its DNA methylation status of its promoter domain.

METHODThe expression of WT1 gene and its DNA methylation status were assayed in leukemia cell lines and normal peripheral blood mononuclear cells (PBMNC) by RT-PCR and MS-PCR.

RESULTSWT1 was overexpressed in HL60, K562 and KG1 leukemia cell lines, but not in U937 and PBMNC. Methylation of WT1 promoter was not observed in HL60 cells.

CONCLUSIONDNA methylation of WT1 gene promotor did not inhibit its expression. Other mechanisms may appear to regulate the WT1 expression.

Cell Line, Tumor ; DNA Methylation ; Genes, Wilms Tumor ; Humans ; Leukemia ; genetics ; Polymerase Chain Reaction ; Promoter Regions, Genetic

LRP16 is a novel gene which was found in our laboratory by using methylation-sensitive restriction landmark genomic scanning (RLGS) technique. In order to clone the full-length cDNA of this leukemia relapse associated gene, the method of rapid amplification of cDNA end (RACE) was employed. By optimizing some procedures of RACE method, the 5'- and 3'-untranslated region of LRP16 cDNA was successfully sequenced. Then, the full length of LRP16 cDNA and open reading frame (ORF) was constructed and was registered in GenBank. The above-mentioned procedure demonstrated RACE technique is a rapid and sensitive method for cloning unknown gene. Especially, it is very useful to cloning the 5'- and 3'-untranslated region of a novel gene.

Objective To investigate the sagittal correction effect and clinical efficacy of multisegment Smith-Peterson osteotomy (SPO) and single-segment pedicle subtraction osteotomy (PSO) in the treatment of old osteoporotic vertebral compression fracture (OVCF)combined with kyphosis.Methods A retrospective case control study was conducted to analyze the clinical data of 24 patients with old OVCF combined with kyphosis admitted to Traditional Chinese Medical Hospital of Xiaoshan from February 2014 to July 2016.There were 10 males and 14 females,aged 58-72 years,with an average of 65.6 years.Thirteen patients were treated with multi-segment SPO (Group A),six of whom underwent two-segment SPO and seven underwent three-segment SPO.Eleven patients were treated with single-segment PSO (Group B).The operation time,intraoperative bleeding volume,the number of cases using cementreinforced nail track,postoperative drainage,hospitalization time and postoperative complications were compared between the two groups.Cobb angle,sagittal vertical axis (SVA),thoracic kyphosis angle (TK) and lumbar lordosis angle (LL) of kyphosis deformity were measured before operation,after operation and at the last follow-up.Oswestry Dysfunction Index (ODI) and Visual Analogue Scale (VAS) were used to evaluate the clinical efficacy at the last follow-up.Results All patients were followed up for 6-24 months,with an average of 15.4 months.The operation time of Group A was (198.1 ± 27.3)minutes,while that of Group B was (237.6 ± 36.1)minutes (P ＜ 0.05).The amount of intraoperative bleeding in Group A was (1 256.2 ± 389.4) ml,while that in Group B was (1 525.6 ±457.1)ml (P ＜ 0.05).Two patients in Group A and five patients in Group B were treated with cement-reinforced nail track (P ＜ 0.05).There was no significant difference in drainage and hospitalization time between the two groups (P ＞ 0.05).Three patients in Group A and one patient in Group B had cerebrospinal fluid leakage (P ＜ 0.05).The postoperative SVA was (1.4 ± 0.7) cm in Group A and (-1.1 ± 0.6) cm in Group B (P ＜ 0.05).No significant differences were found between the two groups in Cobb angle,TK and LL of kyphosis (P ＞ 0.05).There was no significant difference in SVA,Cobb angle,TK and LL between the two groups at the last follow-up (P ＞ 0.05).There was no significant difference in ODI and VAS between the two groups after operation and at the last follow-up (P ＞ 0.05).No complications such as spinal cord injury,embolism caused by cement leakage,extraction or rupture of pedicle screw occurred in either group.Conclusion For old osteoporotic vertebrae compressed fractures combined with kyphosis,multi-segment SPO and single-segment PSO can achieve good sagittal correction and clinical efficacy.Single-segment PSO has better correction effect and lower incidence of cerebrospinal fluid leakage,yet accompanied with disadvantages of overcorrection,screw loosening,longer operation time and more blood loss.

Objective To understand the delay factors of the treatment and diagnosis among pulmonary tuberculosis patients, and to provide scientific evidence for tuberculosis control.Methods A total of 128 pulmonary tuberculosis patients who was newly diagnosed and treated in Xiaoshan were enrolled in this study.The data of demography,medical history and tuberculosis knowledge were collected through questionnaires.Statistical method was used to analyze the current situation and delay factors of the treatment and diagnosis.Results The rate of treatment delay was 25.00%.Female patients without hemoptysis symptom,maintenance elderly,little knowledge of tuberculosis and low degree of education were relative to treatment delay among pulmonary tuberculosis patients (P <0.05 ).Multivariate logistic regression analysis showed that female(OR =6.39,95%CI:1.66 -24.61),was the delay factors of treatment.The rate of diagnosis delay was 50.78%.Multivariate logistic regression analysis showed that preliminary treatment in village or private clinics (OR =2.10,95%CI:1.00 -4.39),preliminary treatment without X ray (OR =3.91,95%CI:1.16 -13.19)and maintenance elderly (OR =57.43,95%CI:4.43 -744.30)were risk factors for delayed diagnosis.Conclusion The influencing factor for treatment and diagnosis delay are female,preliminary treatment in village or private clinics,preliminary treatment without X ray and maintenance elderly.

OBJECTIVETo study the role of PCR technique in detection of mycobacterium tuberculosis in the samples from joint tuberculosis, and to evaluate the clinical value of PCR in diagnosis of joint tuberculosis.

METHODSFrom June 1993 to August 2001, PCR was used to detect DNA of mycobacterium tuberculosis, and the standard culture was applied to detect mycobacterium tuberculosis. Mycobacterium tuberculosis were respectively blindly by the two techniques in the samples obtained from 95 patients with joint tuberculosis (55 males and 40 females, the age ranging from 2 to 75 years, with an average of 34 years). The positive rate of mycobacterium tuberculosis detection was calculated.

RESULTSIn the detection of mycobacterium tuberculosis, positive rate was 82% (78/95) in PCR technique, and 16% (15/95) in standard culture technique. There were statistical differences between the two groups (chi2=67, P<0.001). The whole process of PCR amplification was automatic and could be finished within several hours, and the detecting time was considerably shorter.

CONCLUSIONPCR technique is a rapid, simple, sensitive and specific method for detection of mycobacterium tuberculosis in the samples of joint tuberculosis, showing more marked advantages than the standard culture technique. It is valuable in the early rapid diagnosis and differential diagnosis of joint tuberculosis.

Adolescent ; Aged ; Child ; Child, Preschool ; Culture Techniques ; methods ; Female ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; Polymerase Chain Reaction ; methods ; Tuberculosis, Osteoarticular ; diagnosis ; microbiology ; Young Adult

OBJECTIVETo investigate the effect of Coriolus versicolor polysaccharide B (CVP-B) on increased membrane glycosaminoglycans (GAG) expression and intracellular glutathione (GSH) of RAW264.7 macrophages exposed to angiotensin II (Ang II).

METHODSThe plasma membrane of RAW264.7 macrophages exposed to Ang II treatment was isolated by ultracentrifugation, and the membrane GAG expression was analyzed using 1, 9-dimethylmethylene blue (DMMB) spectrophotometric assay for sulfated GAG. The intracellular reduced GSH was determined using fluorophotometry.

RESULTSThe GAG content in the macrophage membranes increased by up to 54% following cell exposure to 1.0 micromol/L Ang II, whereas in presence of 1.0 micromol;/L Ang II, CVP-B at 1, 10, and 50 microg/ml decreased the GAG content by 13%, 43% (P<0.01), and 52% (P<0.01), respectively. The macrophage GSH activity decreased by 69% following incubation with 1.0 micromol;/L Ang II for 24 h, and CVP-B treatment at 1, 10, and 50 microg/ml in presence of 1.0 micromol;/L Ang II resulted in significant increment of GSH activity by 31%(P<0.05), 104% (P<0.01), and 168% (P<0.01), respectively.

CONCLUSIONThese data provide the first evidence that CVP-B inhibits elevated GAG expression in RAW264.7 macrophage membrane induced by Ang II.

Agaricales ; chemistry ; Angiotensin II ; pharmacology ; Animals ; Cell Line ; Cell Membrane ; metabolism ; Glutathione ; analysis ; Glycosaminoglycans ; analysis ; Macrophages ; metabolism ; Mice ; Polysaccharides ; pharmacology

OBJECTIVETo analyze the pelvic stability after type I resection of iliac tumor.

METHODSSix adult cadaveric specimens were tested. The iliac subtotal resection models were established according to Ennecking's type I resection. Markers were affixed to the key region of the pelves. Axial loading from the proximal lumbar was applied by MTS load cell in the gradient of 0-500 N in the double feet standing state. Images in front view were obtained using CCD camera. Based on Image J software, displacements of the first sacral vertebrae (S1) of the resected pelves and the intact pelves were calculated using digital marker tracing method with center-of-mass algorithm.

RESULTSSerious instabilities were found in the resected pelves. S1 rotational movements around the normal side femoral head of the resected pelvis were found. The average vertical displacement of S1 of the resected pelvis was (7 +/- 3) mm under vertical load of 500 newtons, which were 8.3 times compared to the intact pelvis. The average angle of S1 rotation around the normal side femoral head of the resected pelvis was (4.0 +/- 1.8) degrees, which were 12.5 times compared to the intact pelvis.

CONCLUSIONSBiomechanical model of type I resection of iliac tumor are established. Essential pelvic reconstruction must be introduced because of the serious instability of the bone defection after tumor resection.

Adult ; Aged ; Biomechanical Phenomena ; Bone Neoplasms ; physiopathology ; surgery ; Female ; Humans ; Ilium ; injuries ; Male ; Middle Aged ; Models, Biological ; Pelvis ; physiopathology ; Range of Motion, Articular

Aim To investigate the impact of Cinobu-facini on the proliferation,invasion,and apoptosis of HepG2 cells and the underlying mechanism.Methods The proliferation of HepG2 cells was assessed using the CCK-8 method following treatment with Cinobufaci-ni.The invasion capability of HepG2 cells was evalua-ted through Transwell assay after exposure to Cinobufa-cini.The apoptosis rates of HepG2 cells post Cinobufa-cini intervention were measured using flow cytometry,and the expression levels of VEGF in the culture medi-um of HepG2 cells were determined using enzyme-linked immunoassay.Furthermore,qRT-PCR and Western blot analyses were conducted to assess the im-pact of Cinobufacini on mRNA and protein expression levels related to the CXCL5/FOXD1/VEGF pathway.The interaction between CXCL5 and FOXD1 was inves-tigated via co-immunoprecipitation.Results Cinobufa-cini treatment led to a gradual decrease in HepG2 cell viability in a dose-dependent manner compared to the control group(P＜0.05).Moreover,Cinobufacini sig-nificantly suppressed HepG2 cell invasion(P＜0.05)while enhancing cell apoptosis(P＜0.05).Notably,Cinobufacini exhibited inhibitory effects on the CX-CL5/FOXD1/VEGF pathway,as evidenced by re-duced expression of related mRNA and proteins(P＜0.05).FOXD1 was identified as the binding site of CXCL5.Overexpression of CXCL5 resulted in in-creased proliferation and VEGF secretion by HepG2 cells(P＜0.05),and increased expression of FOXD1 and VEGF(P＜0.05).However,Cinobufacini inter-vention effectively inhibited liver cancer cell prolifera-tion and invasion(P＜0.05),promoted apoptosis(P＜0.05),reduced VEGF secretion by HepG2 cells(P＜0.05),and downregulated the expression of CXCL5 and FOXD1 in HepG2 cells(P＜0.05);but com-pared with the unexpressed group of Cinobufacini,its ability to inhibit cell activity was weakened(P＜0.05),and its ability to inhibit the expression of CX-CL5,FOXD1,and VEGF was weakened(P＜0.05).Conclusion Cinobufacini may inhibit HepG2 cell pro-liferation and invasion and promote HepG2 cell apopto-sis by regulating the CXCL5/FOXD1/VEGF pathway.

OBJECTIVETo develop a chemiluminescent enzyme-linked immunosorbent assay (CLEIA) for the detection of HTNV IgM antibody.

METHODSBlack solid 96 well microplate was coated with anti-human IgM-microantibody, HRP labeled HTNV recombinant nucleotide antigen was used as detection antigen, luminol-H2O2 was used as substrate, a CLEIA was established for the detection of HFRS patient serum IgM antibody and comparison of detection sensitivity, specificity, and stability were made between CLEIA and MacELISA.

RESULTSCorrelate coefficient of CLEIA with MacELISA is 0.97; detection sensitivity of CLEIA is 100 percent while that of MacELISA is 92.1 percent; detection specificity of CLEIA and MacELISA are both 100 percent; coefficient of variance for intra-assay and inter-assay of CLEIA are both less than 15 percent, which are comparative with MacELISA.

CONCLUSIONThe established method of CLEIA is a sensitive, selective, and stable method; it is suitable for the early detection of HFRS patient serum IgM antibody.

Antibodies, Viral ; blood ; Antibody Specificity ; Enzyme-Linked Immunosorbent Assay ; methods ; Hantavirus ; immunology ; Hemorrhagic Fever with Renal Syndrome ; immunology ; Humans ; Immunoglobulin M ; blood ; Luminescent Measurements ; methods

BACKGROUNDEchinococcosis, coenurosis and cysticercosis are debilitating diseases which prevail in China. Immunological diagnosis of metacestodosis is important in disease control. The 8-kDa glycoproteins from taeniid cestodes have successfully been used for diagnosis of human cysticercosis in immunological assays. The aim of the present study was to investigate genetic variations and phylogenetic relationships of the 8-kDa proteins for evaluating the possibility of utilizing these proteins as diagnostic antigens for other metacestode infections.

METHODSThe genes and complementary DNAs (cDNAs) encoding the 8-kDa proteins from Echinococcus (E.) granulosus, Taenia (T.) multiceps and T. hydatigena were amplified using PCR method. Their amplicons were cloned into the vector pMD18 and the positive clones were sequenced. Sequence data were analyzed with the SeqMan program, and sequence homology searches were performed using the BLAST program. Alignments were conducted using the ClustalX program, and the phylogenetic analyses were performed with the Protein Sequences Program and the Puzzle Program using the Neighbor-joining method.

RESULTSFifteen, 18 and 22 different genomic DNA sequences were identified as members of the 8-kDa protein gene family from E. granulosus, T. multiceps and T. hydatigena, respectively. Eight, four and six different cDNA clones respectively from E. granulosus, T. multiceps and T. hydatigena were characterized. Analysis of these sequences revealed 54 unique 8-kDa protein sequences. Phylogenetic trees demonstrated that the taeniid 8-kDa proteins are clustered into eight clades at least: Ts18, Ts14, TsRS1, TsRS2, T8kDa-1, T8kDa-2, T8kDa-3 and T8kDa-4.

CONCLUSIONWe found that the gene family encoding for the taeniid 8-kDa antigens is comprised of many members with high diversity, which will provide molecular evidence for cross-reaction or specific reaction among metacestode infections and may contribute to the development of promising immunological methods for diagnosis of metacestodosis.

Amino Acid Sequence ; Animals ; DNA, Helminth ; genetics ; Echinococcus granulosus ; genetics ; metabolism ; Genetic Variation ; genetics ; Glycoproteins ; chemistry ; classification ; genetics ; Helminth Proteins ; chemistry ; classification ; genetics ; Molecular Sequence Data ; Phylogeny ; Sequence Homology, Amino Acid ; Taenia ; genetics ; metabolism