Objective Myelodysplastic spndrome (MDS) patients' chromosome aberrations were detected by CC and FISH method respectively,to establish standard FISH platform of diagnosis of MDS,to compare the differences of FISH and CC in the detection of MDS chromosome aberrations.Methods Chinese MDS patients fulfilled with the Vienna minimum diagnostic criteria were tested by CC and FISH method,and the patients' clinical data was collected at the same time.Results The standard FISH platform of diagnosis of MDS was established successfully,including probe combination of-5/5q-,+8,-7/7q-,20q-,-Y,the success rate was 100 ％.In 83 cases of MDS,chromosome aberrations rate detected by two methods was 42.2 ％,30.1％ of CC method and 33.7 ％ of FISH method,the accordant diagnostic rate was 76.1％.FISH had higher positive rate than CC method (29.6 ％ vs 18.6 ％,P =0.049) in the IPSS intermedian-risk 1 group,but lower positive rate in IPSS intermedian-risk 2 group (62.5 ％ vs 81.2 ％,P =0.036).There were 11 (19.0 ％) cases of clonal chromosome aberrations detected by FISH method but not detected by CC method,in which 10 (90.9 ％) cases were intermedian-risk 1 group (IPSS grouping).Evaluate IPSS by FISH and CC respectively,the accordant rate was 86.7 ％.Evaluate IPSS by CC alone will result in 3 patients (3.6 ％)decline in risk stratification,and evaluate IPSS by FISH alone will result in 8 patients (9.6 ％) decline in risk stratification.There were 35 (42.2 ％) cases of chromosome aberrations detected by the two methods,including 13 (15.7 ％) cases of complex karyotype and 22 (26.5 ％) cases of single abnormal karyotype.The most common chromosomal aberration was +8,a total of 9 (10.8 ％) cases,followed by abnormalities of chromosome 7,a total of 7 (8.4 ％) cases.Conclusion The study has established standard FISH platform of diagnosis of MDS,the method is stable,sensitive and rapid.FISH technology joint CC technology can improve the patients with MDS chromosome aberration detection rate.Compared to CC,FISH technique can improve the detection rate of the intermedian-risk 1 IPSS grouping MDS patients.Because of limited probe,FISH technique can not entirely replace the role of CC method in MDS,but can complement each other.

AIMTo set up an IR-HIRc cell model for screening the inhibitor of GFAT (glutamine: fructose-6-phosphate amidotransferase) , the key enzyme in the hexosamine biosynthesis pathway (HBP).

METHODSFor GFAT activity assay, the GDH method was improved by adjusting the value of pH in the reaction system and the concentrations of the reactants. The sensitivity to insulin in the cells was estimated by the measurement of insulin-induced glucose-uptake. The IR-HIRc model was set up by the stimulation of long-action insulin for 36 h. The IR-HIRc model and GDH method was used for screening GFAT inhibitor.

RESULTSWith the administration of 25 nmol x L(-1) long-action insulin in HIRe cells for 36 hours, the GFAT activity increased by 47% and the insulin-induced glucose-uptake decreased by 21%. Azaserine, a GFAT inhibitor, inhibited GFAT activity significantly in a dose-dependent manner in IR-HIRc model.

CONCLUSIONWith the stimulation of 25 nmol x L(-1) long-action insulin for 36 h, excess hexosamine flux and insulin resistant in IR-HIRc cell model was set up, which can be used for screening

Animals ; Azaserine ; administration & dosage ; pharmacology ; Cell Line ; Dose-Response Relationship, Drug ; Fibroblasts ; cytology ; metabolism ; Glucose ; metabolism ; Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) ; antagonists & inhibitors ; metabolism ; Hexosamines ; biosynthesis ; Insulin ; pharmacology ; Insulin Resistance ; Models, Biological ; Rats ; Recombinant Proteins ; metabolism

AIMTo explore the influence of acute hypoxia and intermittent hypoxic acclimatization on vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1alpha (HIF-1alpha) gene expression in HepG2 cells underlying their possible biological significance.

METHODSHepG2 was cultured in 1% O2 for 24 hours, then in 21% O2 for another 24 hours, which composed a hypoxic exposure cycle. After 6 cycles, HepG2 cells reached the status of hypoxic acclimatization. Gene transcription and translation of VEGF and HIF-1alpha were detected with Northern blot and Western blot methods.

RESULTSAcute hypoxia could induce gene transcription and translation of VEGF and HIF-1alpha. After intermittent hypoxia acclimatization, the contents of VEGF and HIF-1alpha mRNA were 108.6% +/- 17.7% and 116.7% +/- 19.8% of those in normoxic control cells, while the protein contents were significantly increased to 1.4 and 2.7 times of those in control cells, respectively (P < 0.05). The protein expression levels of VEGF and HIF-1alpha were decreased in cells subjected to hypoxia acclimatization compared to cells treated with acute hypoxia.

CONCLUSIONWhen HepG2 cells reached the status of hypoxic acclimatization, the acute hypoxia-induced increment of VEGF gene transcription and translation in cells were inhibited, in which HIF-1alpha might play an important role.

Acclimatization ; genetics ; Cell Hypoxia ; genetics ; Gene Expression ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Nuclear Proteins ; genetics ; Oxygen ; metabolism ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Objective To investigate the association between the single nucleotide polymorphism (SNP) of leukotriene C4 synthase(LTC4S) rs730012 in the childhood asthma.Methods Sequence specific primers-polymerase chain reaction was used to assess the genetic polymorphism of LTC4S rs730012 in 105 asthma children with different order of severity and 128 non-asthma children in our hospital in the northeast of China to analyse the association between the SNP of LTC4S rs730012 and susceptibility,clinical phenotype in asthma children.Results (1) In case group,genotype frequencies of A/C,A/A and C/C were 71.4％ 、25.7％ 、2.9％,allele frequencies for A,C were 84.3％,15.7％.In control group,the genotype frequencies of A/A,A/C,C/C,were 70.3％,28.9％,0.8％,allele frequencies for A,C were 84.8％,15.2％.No significant difference was found in AA genotype and C allele frequencies between case and control grouP(x2 =0.035、0.020,P both ＞0.05).(2) C/C genotype or C allele frequencies in moderate-severe asthma group were significantly higher than the mild asthma group(x2 =5.859、5.641,P both ＜ 0.05);(3) SaO2 of A/A group was significantly higher than A/C and C/C group (t =2.976,Pboth ＜ 0.05),and FeNO and obstructive ventilatory disorder incidence rate in A/C,C/C group were higher than A/A group,the differences were statistically significant (t =2.946、x2 =5.564,P both ＜ 0.05).Conclusion The SNP of LTC4S rs730012 is associated with the order of severity,SaO2,FeNO,pulmonary function in asthma children of northeast China.However,the rs730012 is not associated with the susceptibility for asthma.

Objective:To observe the effect of electroacupuncture (EA) on the expressions of acetylcholine (ACh) and mucin 5AC (MUC5AC) in the lungs of rats with chronic obstructive pulmonary disease (COPD),and explore the mechanism of EA in treating COPD.Methods:Thirty Sprague-Dawley (SD) rats were randomly divided into a control group,a COPD group,and an EA group,with 10 rats in each group.The control group was a group of normal rats.The COPD rat model was induced by cigarette smoke combined with lipopolysaccharide (LPS).The COPD rats were treated with EA at bilateral Feishu (BL 13) and Zusanli (ST 36) in the EA group,30 min each time,once a day,successively for 14 d.The lung function was tested.The contents of ACh and MUC5AC in lungs and bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA).Pearson method was used to analyze the correlation between pulmonary function and the content of MUC5AC in lungs.The mRNA and protein expressions of MUC5AC in lung tissues were detected by real-time polymerase chain reaction (RT-PCR) and Western blot (WB),respectively.The immune response of MUC5AC was observed by immunohistochemistry.Results:Eight rats were left in each group,and the other two died.Compared with the control group,the total airway resistance (Raw) increased significantly and dynamic compliance (Cdyn) decreased significantly in the COPD group (P＜0.01);compared with the COPD group,the Raw level declined significantly and Cdyn increased significantly in the EA group (P＜0.01).The contents of ACh and MUC5AC in the lungs and BALF were remarkably higher in the COPD group compared with those in the control group (P＜0.01,P＜0.001);compared with the COPD group,the contents of ACh and MUC5AC were significantly lower in the EA group (P＜0.05,P＜0.001).There was a negative correlation between MUC5AC content and lung function (P＜0.001).The mRNA and protein expressions of MUC5AC in the lungs were significantly higher in the COPD group than in the control group (P＜0.001);compared with the COPD group,the expressions were significantly lower in the EA group (P＜0.01).Compared with the control group,the immune response of MUC5AC in the airway epithelium significantly increased in the COPD group (P＜0.001);the immune response of MUC5AC was significantly lower in the EA group compared with that in the COPD group (P＜0.001).Conclusion:EA treatment can improve the lung function of COPD rats,which may be related to its effect in the down-regulation of ACh and MUC5AC contents in the lungs as well as the inhibition of mucus hypersecretion.

PURPOSE: Five members of the zinc finger of the cerebellum (ZIC) family—ZIC1, ZIC2, ZIC3, ZIC4, and ZIC5—have been shown to be involved in various carcinomas. Here, we aimed to explore the clinicopathologic and prognostic roles of ZIC family members in invasive breast cancer patients using immunohistochemical analysis, western blotting analysis, and real-time quantitative polymerase chain reaction (RT-qPCR). METHODS: A total of 241 female invasive breast cancer patients who underwent radical mastectomy between 2009 and 2011 were enrolled. ZIC proteins in 241 pairs of breast tumors and corresponding normal tissues were investigated using immunohistochemistry and the clinicopathologic roles of proteins were analyzed using Pearson's chi-square test. Kaplan-Meier curves and Cox regression analysis were also used to analyze the prognostic value of the ZIC proteins. In addition, 12 pairs of fresh-frozen breast tumors and matched normal tissues were used in the western blotting analysis and RT-qPCR. RESULTS: Only ZIC1 expression in normal tissues was obviously higher than that in tumors (p < 0.001). On multivariate analysis, ZIC1 expression (in overall survival analysis: hazard ratio [HR], 0.405, 95% confidence interval [CI], 0.233–0.702, p=0.001; in disease-free survival analysis: HR, 0.395, 95% CI, 0.234–0.669, p=0.001) was identified as a prognostic indicator of invasive breast cancer. CONCLUSION: ZIC1, but not the other proteins, was obviously decreased in breast tumors and associated with clinicopathologic factors. Thus, ZIC1 might be a novel indicator to predict the overall and disease-free survival of invasive breast cancer patients.
Blotting, Western ; Breast Neoplasms* ; Breast* ; Cerebellum* ; Disease-Free Survival ; Female ; Humans ; Immunohistochemistry ; Mastectomy, Radical ; Multivariate Analysis ; Pathology ; Polymerase Chain Reaction ; Prognosis ; Zinc Fingers* ; Zinc*

OBJECTIVETo investigate the correlation of vascular endothelial growth factor D (VEGF-D) expression, microlymphatic density (MLD) and microvessel density (MVD) levels with the development and metastasis of rectal cancer.

METHODSEighty specimens from resected middle-lower rectal cancer diagnosed by pathology were examined by immunohistochemistry for VEGF-D,MLD and MVD. Simultaneously, 40 biopsy specimens from rectal polyps and 80 specimens from normal rectal tissue were examined as controls. Correlation between the expression of above three factors and the tumor size, gross morphology, histological type, metastasis, differentiation grade, infiltration depth, Dukes stage, lymph node metastasis and long-distance metastasis before operation were investigated with Spearman method.

RESULTS(1) Positive expression rate of VEGF-D was 55 % (44/80) in rectal cancer, and zero in rectal polyps and normal rectal tissues. The expression of VEGF-D in rectal cancer was significantly higher than that in rectal polyps and normal rectal tissues(P<0.05). MLD was significantly higher in rectal cancer (2.80+/-1.31) than that in rectal polyps (0.50+/-0.72) and normal rectal tissues(0.25+/-0.44)(P<0.05).Meanwhile MVD was significantly higher in rectal cancer (80.10+/-23.18) than that in rectal polyps (27.00+/-11.01) and normal rectal tissues (10.45+/-5.34) (P<0.05). (2) VEGF-D, MLD and MVD were positively correlated with lymph node metastasis and long-distance metastasis before operation (P<0.05). (3) VEGF-D was positively correlated with MLD (P<0.05) and MLD was positively correlated with MVD as well(P<0.05).

CONCLUSIONSLymphangiogenesis exists in rectal cancer tissues. VEGF-D and MLD can be used as good predictors of lymphangiogenesis and they are the important factors affecting biological behavior of rectal cancer. Lymphangiogenesis and angiogenesis may have a cooperative function in the development of rectal cancer.

Female ; Humans ; Lymphangiogenesis ; Lymphatic Metastasis ; Male ; Microcirculation ; Microvessels ; pathology ; Middle Aged ; Neoplasm Staging ; Neovascularization, Pathologic ; pathology ; Rectal Neoplasms ; blood supply ; pathology ; Vascular Endothelial Growth Factor D ; genetics ; metabolism

OBJECTIVETo study whether dendritic cells (DCs) derived from the peripheral blood in chronic hepatitis B patients can induce specific T cell immune response.

METHODS(1)The subjects were divided into 3 groups: chronic hepatitis B group (CHB), acute hepatitis B group (AHB), and normal donor group (ND). The peripheral blood mononuclear cells (PBMCs) isolated from those subjects were stimulated with HBcAg 18 to 27 CTL epitope peptide, and intracellular cytokine staining (ICCS) was used for detecting IFN-gamma, IL-2 and TNF-alpha produced by CD8+ T cell. (2) DCs generated from PBMCs were pulsed with HBcAg 18 to 27 CTL epitope peptide, then were cocultured with autologous lymphocytes for 10 days to induce antigen-specific T cell, which was assessed by ICCS and cytotoxic assay.

RESULTS(1) The memory effect of the PBMCs from AHB group to HBcAg 18 to 27 CTL epitope peptide was stronger than that from CHB or ND group (t=2.508-3.305, P<0.05). (2)After lymphocytes were cocultured with DC treated with HBcAg 18 to 27 CTL epitope peptide, antigen-specific T cell effect was induced. And the killing rates were (57.0+/-23.0)%, (49.5+/-20.2)%, (21.8+/-12.9)% at the effector/target of 30:1, 10:1, 3:1, which were higher than that in control group.

CONCLUSIONSThe memory T cells against HBV antigen lacks in CHB patients. DCs from CHB patients pulsed with HBcAg 18 to 27 epitope peptide can induce HBV antigen-specific T cell, which can kill specific target cells and produce cytokines involved in virus clearance.

Adult ; CD8-Positive T-Lymphocytes ; immunology ; Cells, Cultured ; Dendritic Cells ; drug effects ; immunology ; virology ; Epitopes, T-Lymphocyte ; immunology ; Female ; Hepatitis B Core Antigens ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Leukocytes, Mononuclear ; immunology ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; immunology

OBJECTIVESTo dynamically observe how glioma stem cells promote the tumor formation and angiogenesis, and to study the correlation between the distribution of glioma stem cells and microvessels within different growth stages of subcutaneous tumor.

METHODSStem cell medium culture and magnetic activated cell sorting were carried out to obtain CD133+ cells from C6 rat glioma cell line. Sprague Dawley (SD) rat ears model were established to observe glioma stem cells promoting blood vessel formation. Subcutaneous glioma model of C6 and immunohistochemical staining of hypoxia inducible factor-1α (HIF-1α) and CD133 were used to investigate the relationship between distribution of glioma stem cells and microvessels. Expressions of CD133 protein in each stage of the subcutaneous tumor were detected by Western blot.

RESULTSIsolation and identification of glioma stem cells deprived from C6 glioma cell line successfully, the establishment of ears model showed real-time dynamic observation of CD133+ cells involved in angiogenesis and tumor formation. SD rat model of subcutaneous glioma showed the initial of tumor formation, CD133+ cells scattered. With tumor growth, CD133+ cells began to tend to capillaries, in late distributed clusters in perivascular. Meanwhile as tumor growth, CD133 protein expression was gradually increased: the values of Western blot analysis of CD133 expression on 6, 9, 12, 15, 20 d were 0.208±0.004, 0.282±0.003, 0.360±0.004, 0.564±0.135, 0.756±0.007, the differences were significant between different groups (F=2601.681, P<0.01). At a high magnification, the CD133 scores with immunohistochemical staining on 6, 9, 12, 15 d were 0.8±0.4, 2.4±0.5, 4.0 ± 0.7, 6.0±0.7; HIF-1α scores were 0.8±0.4, 2.8±0.8, 5.0±0.7, 6.8±0.4. By Spearman rank correlation analysis found that the relationship between CD133 and HIF-1α expression was positively correlated (r=0.921, P<0.01).

CONCLUSIONSGlioma stem cells promote angiogenesis more than non-stem cells; HIF-1α and its downstream gene product might mediate the distribution of glioma stem cells around the perivascular.

Animals ; Cell Line, Tumor ; Glioma ; blood supply ; metabolism ; pathology ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Microvessels ; pathology ; Neoplasm Transplantation ; Neoplastic Stem Cells ; pathology ; Neovascularization, Pathologic ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the correlation between impaired non-viral specific immune function of dendritic cell (DC) and viral clearance and cytotoxic T lymphocyte (CTL) response to HBV or HCV in patients with HBV and HCV coinfection.

METHODSTwenty-five patients with HBV and HCV coinfection were investigated in this study. In 1994 and 2002, biochemical and virological markers and quantitative serum HBV DNA and HCV RNA levels were detected in these patients. According to the virus clearance status, these patients were divided into 4 groups: 14 patients with both HBV and HCV clearance (Group A), 6 patients with HCV clearance only (Group B), 3 patients with HBV clearance only (Group C), and 2 patients with persistent infection of HBV and HCV (Group D). Phenotypes and immune functions of monocyte-derived DCs were compared between these groups. 51Cr release assay were used to measure CTL response to epitopes derived from HBV, HCV or influenza virus (as positive control) in HLA-A2+ patients.

RESULTSImpaired non-viral specific immune functions of DCs were observed in group B, C and D compared with group A and normal donors (Group N). These impaired functions included CD86 decreasing expression and lower capacity to stimulating allogenic T cells and uptaking antigen. The specific CTL response to HBV- and HCV-derived peptides could be induced in group A (12/12). The specific CTL response to HBV-derived peptides or to HCV-derived peptides could be induced in group C (3/3) or B (5/5), respectively. But the specific CTL response to both of two HBV-derived peptides or two HCV-derived peptides could not be induced in group C (0/3) or B (0/5), respectively. And no CTL response to HBV or HCV-derived peptides could be induced in groups D (0/1) and N (0/4).

CONCLUSION1. The results suggest that specific CTL response to HBV or HCV play a vital role in the viral clearance. 2. The DCs with impaired non-viral specific immune functions exist in chronic patients with HBV and/or HCV infection, but do not interfere with clearance and CTL response to HBV or HCV. It is reasonable to speculate that impaired functions of DCs result from viral infection.

Adult ; Dendritic Cells ; immunology ; Female ; Hepacivirus ; immunology ; Hepatitis B virus ; immunology ; Humans ; Immunophenotyping ; Lymphocyte Culture Test, Mixed ; Male ; Middle Aged ; T-Lymphocytes, Cytotoxic ; immunology