Objective To study the effects of miR-200b on proliferation and migration of sw620 colon cancer cells,and its regulation effect on E-cadherin expression.Methods The expressions of miR-200b in sw620 cells at 24 h and 72 h after pEGP-miR-200b transfection were detected by real-time PCR (RT-PCR).The change of the expression level of E-cadherin after miR-200b transfection was detected using the methods of RT-PCR and Western blot.The proliferation and migration abilities were measured by MTT and scratch test after miR-200b transfection.Results The expressions of miR-200b in sw620 cells at 24 h and 72 h after pEGP-miR-200b transfection raised significantly compared to the control group (t =11.579,P ＜ 0.01 ; t =11.579,P ＜0.01).MiR-200b transfection inhibited the proliferation abilities of sw620 cells.It is the most significant of the inhibitory effect on the third day and the inhibition rate was 55.34％.MiR-200b transfection markedly inhibited the migration abilities of sw620 cells.The two groups had significant difference in the migration distance of 24,48,72 h (t =11.579,P ＜0.01 ; t =10.419,P ＜0.01 ; t =6.955,P ＜0.01).The mRNA and protein expressions of E-cadherin gene increased significantly by transfecting miR-200b gene in sw620 cells (t =10.432,P ＜ 0.01 ; t =8.325,P ＜ 0.O1).Conclusion Up-regulated expression of miR-200b could inhibite the proliferation and migration abilities of sw620 colon cancer cells.The involved molecular mechanism is probably related to the change of E-cadherin expression.

OBJECTIVETo explore the methylation status in promoter region of norepinephrine transporter gene (NET, SLC6A2) in heart failure ( HF) patients and its correlation with qi deficiency/blood stasis syndrome (QDS/BSS).

METHODSThirty-six patients with heart failure (NYHA classification III to IV) were recruited in the study (as the heart failure group) and their scores of QDS/BSS were evaluated. Besides, a healthy elderly group (30 cases) and a healthy youth group (30 cases) were also set up. They were recruited from Physical Examination Center of Fujian Provincial Hospital. Pyrosequencing was applied to detect the methylation in promoter region of SLC6A2 gene, and the total methylation index (MTI) of CpG island was calculated. The correlation between the methylation status in promoter region of SLC6A2 and scores of QDS/BSS was assessed using Pearson and Partial analyses. Risk factors were screened and adjusted using Logistic regression.

RESULTSBy one-factor analysis of variance, the total MTI in the HF group (219.72% ± 54.03%) was obviously higher than that in the healthy elderly group (194.47% ± 34.92%) and the healthy youth group (161.60% ± 41.11%) (all P < 0.05). Meanwhile, the total MTI was higher in the healthy elderly group than in the healthy youth group (P < 0.01). By covariance analysis , after controlling age and BMI, the total MTI was higher in the HF group than in the healthy elderly group (P = 0.041), while it was higher in the healthy elderly group than in the healthy youth group (P = 0.016). Age was found to play an essential role in affecting MTI of SLC6A2 gene promoter region among the 3 groups (F = 16.447, P = 0.01). The total MTI was quite lower in the healthy youth group. Results of Partial correlation analysis showed MTI was positively correlated with scores of qi deficiency and blood stasis respectively (r = 0.494 and 0.419 respectively, both P < 0.05). Logistic regression analysis showed after adjusting confounding factors, the relative risk (OR value) of total MTI of SLC6A2 gene in promoter region was 1.038 (95% CI, 1.006 to 1.071, P = 0.020).

CONCLUSIONSAbnormally elevated methylation of the promoter region of SLC6A2 gene is one of risk factors for HF. In addition, the degree of methylation of the promoter region of SLC6A2 gene was positively correlated with the severity of QDS/BSS.

Adolescent ; Aged ; DNA Methylation ; Heart Failure ; genetics ; physiopathology ; Humans ; Logistic Models ; Medicine, Chinese Traditional ; Norepinephrine Plasma Membrane Transport Proteins ; genetics ; Promoter Regions, Genetic ; Qi

Background: Mast cell activation is a characteristic of irritable bowel syndrome (IBS).Study on mast cell and the related inflammatory mediators in colonic mucosa is helpful for the evaluation and treatment of IBS.Aims: To assess the effect of mesalazine combined with trimebutine on colonic mucosal mast cell and related inflammatory mediators in patients with IBS.Methods: Forty patients with diarrhea-predominant IBS (IBS-D) and 40 patients with constipation-predominant IBS (IBS-C) from Oct.2014 to June 2016 at Shanghai Jiading District Central Hospital were enrolled, 20 healthy volunteers were served as controls.Forty patients with IBS-D and 40 patients with IBS-C were randomly divided into mesalazine+trimebutine group and trimebutine group, the treatment courses were all 4 weeks.Number of mast cell was counted by modified toluidine blue staining.Score of related inflammatory mediators were evaluated by immunohistochemistry.Clinical efficacy was assessed.Results: Compared with healthy controls, number of mast cell at baseline was significantly increased both in IBS-D and IBS-C patients (P<0.05).After treatment with mesalazine+trimebutine, number of mast cell was significantly decreased (P<0.05).At baseline, immunohistochemical staining score of 5-HT, IL-1, TNF-α, histamine, tryptase were significantly increased in IBS patients than in healthy controls (P<0.000 1).After treatment with mesalazine+trimebutine, above-mentioned inflammatory mediators were significantly decreased (P<0.05).In IBS-D patients, the total efficacy rate in mesalazine+trimebutine group was significantly increased than that in trimebutine group (85.0% vs.45.0%, P=0.008).In IBS-C patients, no significant difference in total efficacy rate was found between mesalazine+trimebutine group and trimebutine group (55.0% vs.25.0%, P=0.053).Conclusions: Mesalazine combined with trimebutine is an effective and safe approach to reduce mast cell infiltration and release of related inflammatory mediators, and is more efficient for patients with IBS-D.

OBJECTIVETo investigate the effects of T lymphoma invasion and metastasis inducing factor 1 antisense oligodeoxynucleotides (Tiam 1 ASODN) transfection on the morphology and invasive migration potential of gastric cancer cells.

METHODSThe higher invasive and migratory subgroup (M(H)) were separated from human gastric cancer cell line MKN-45 (M(0)) by laminin adhesion method in vitro. Tiam 1 ASODN was transfected into M(H) cells with liposome, and the expression of Tiam 1 mRNA and protein was determined by RT-PCR and flowcytometry respectively. The changes in morphology, the invasive and migratory potential between Tima 1 ASODN transfected M(H) cells and no transfected M(H) cells were observed by HE stain, cytoskeletal protein stain, scanning electronic microscope (SEM) and Boyden chamber test.

RESULTSCompared with the control, the expression of Tiam 1 mRNA and protein in M(H) cells was significantly decreased after transfected with 0.43 micromol/L ASODN(P< 0.01). The invasive and migratory potential of M(H) cells in vitro was also much more decreased than that of no transfected cells (P< 0.05 or P< 0.01). At the same time, transfected M(H) cells had less membrane surface projections, fewer or shorter pseudopodia, less irregular cytoskeletal network and less spotted-like actin bodys than no transfected M(H) cells did.

CONCLUSIONTiam 1 ASODN transfection can effectively suppress the expression of Tiam 1 in gastric cancer cells and impair its invasive and migratory potential in vitro, which may be fulfilled through modulating the reconstruction of cytoskeleton and decreasing the deforming and migratory potential of gastric cancer cells.

Animals ; Cell Line, Tumor ; Flow Cytometry ; Guanine Nucleotide Exchange Factors ; genetics ; Humans ; Mice ; Mice, Inbred BALB C ; Neoplasm Invasiveness ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; genetics ; Stomach Neoplasms ; genetics ; pathology ; T-Lymphoma Invasion and Metastasis-inducing Protein 1 ; Transfection

Adolescent ; Adult ; Aged ; Bronchiolitis ; complications ; diagnosis ; therapy ; Female ; Humans ; Male ; Middle Aged

OBJECTIVETo study the clinical manifestations and neuroimaging characteristics of pediatric moyamoya disease.

METHODSThe clinical data of 17 children with moyamoya disease were retrospectively studied.

RESULTSThe onset age was between 3 and 14 years. The main clinical manifestations included motor weakness of extremities or hemiplegia, sensory disturbance and headache. Cranial CT or/and MRI examinations predominately showed cerebral infarct. Magnetic resonance angiography (MRA) and digital subtraction angiography (DSA) showed stenosis or occlusion at the terminus of the siphon portions of internal carotid arteries and proximal portions of anterior or middle cerebral arteries, and abnormal vascular networks at the base of brain.

CONCLUSIONSCerebral ischemia is main clinical manifestations in children with moyamoya disease, presenting motor weakness of extremities or hemiplegia, sensory disturbance and headache. DSA is essential to the diagnosis of the disease.

Adolescent ; Angiography, Digital Subtraction ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Humans ; Magnetic Resonance Imaging ; Male ; Moyamoya Disease ; complications ; diagnosis ; therapy ; Tomography, X-Ray Computed

OBJECTIVETo purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.

METHODSThe tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200).

RESULTThe purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained.

CONCLUSIONSWe successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.

Bacterial Proteins ; biosynthesis ; chemistry ; isolation & purification ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; isolation & purification ; Cloning, Molecular ; Crystallization ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Thermoanaerobacter ; enzymology ; genetics ; Transformation, Bacterial

OBJECTIVEN-acetyltransferase 2 (NAT2) and cytochrome P450 2EI (CYP2E1) play a crucial role in the drug metabolic process. The aim of this study was to understand the genotype and phenotype polymorphisms of NAT2 and CYP2E1 in the Han Chinese pediatric population in order to provide a theoretical basis for individualized drug treatment.

METHODSA total of 341 (211 males and 130 females) randomly sampled Han Chinese children, aged from 2 months to 14 years, were enrolled in this study. Genotyping was carried out by PCR method, and metabolic phenotypes were identified.

RESULTSIn this study population, wild genotype was found as a major genotype in seven SNPs of NAT2, rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208 and rs1799931. The frequency of NAT2 fast metabolism was highest (61.3%), followed by middle to slow metabolism (34.1%). Wild genotype also predominated in the four SNPs of CYP2E1 (rs2031920, rs3813867, rs6413432 and rs72559720) named as CYP2E1*5, *6 and *2, with a frequency of 61.3%, 60.1% and 99.4% respectively. As the relationship between CYP2E1 genotype and phenotype was unknown, phenotyping of CYP2E1 was not done.

CONCLUSIONSThe important SNPs of NAT2 and CYP2E1 are predominantly wild genotype in the Han Chinese pediatric population. Fast metabolic phenotype predominates in important SNPs of NAT2.

Adolescent ; Arylamine N-Acetyltransferase ; genetics ; Child ; Child, Preschool ; China ; ethnology ; Cytochrome P-450 CYP2E1 ; genetics ; Female ; Genotype ; Humans ; Infant ; Infant, Newborn ; Male ; Phenotype ; Polymorphism, Single Nucleotide

BACKGROUND:As a kind of undifferentiated precursor cells,the phenotypic differentiation of bone marrow mesenchymal stem cells (BMSCs) remains immaturity,thus it presents weak rejection following transplantation.However,the in vitro directional differentiation of BMSCs into neuronal cells is easy affected by various factors.OBJECTIVE:To observe the immunomodulatory effect and the potential of BMSCs differentiate into neuronal-like cells.DESIGN,TIME AND SETTING:A contrast observation was conducted at the Department of Cytology,Third People's Hospital of Wuxi from January 2008 to March 2009.MATERIALS:Bone marrow was harvested from chips of cancellous or ilium bone dudng hip joint surgery.METHODS:Firstly,the BMSCs were separated and cultured to establish mixed lymphocyte reaction (MLR) system.Secondly,2 samples of peripheral blood mononuclear cells (1×10~5/well) were added into 96-well plate,and then BMSCs treated by mitomycin were added according to different ratios (BMSCs/peripheral blood monouclear cells).At the end,the cells were cultured as follows:Method 1:DMEM+10% fetal calf serum+1 μmol/L RA +20 μg/L basic fibroblast growth factor+20 μg/L epidermal growth factor.Method 2:DMEM+2% dimethyl sulfoxide +100 μmol/L butylated hydroxyanisole.MAIN OUTCOME MEASURES:The growth rate of lymphocyte was measured by ~3H-Thymidine,and the effect of BMSCs on lymphocyte proliferation was observed.Additionally,the differentiation potential of BMSCs into neuronel cells was determined by immunofluorescenca and immunohistochemistrical staining.RESULTS:①The BMSCs inhibited lymphocyte proliferation in MLR system and the influence on proliferation of lymphocyte was direct related to ratio of BMSCs.②Under a light microscope,cytoplasm of BMScs were shrinkd,which presented typical perikaryon morphology at hour 2 after culture with method 1.The majority of BMSCs were formed neuronal-like cells without number changes at hours 3-5,which turned to be dipolar or multipolar neuronal shapes at day 3.There were 60%-70% neuronspecific enolase,45%-50% glial fibrillary acidic protein were positive expressed.However,the positive rate of nidogen was decreased 3.4%.Cells cultured with method 2 became smaller after 2 hours,formed dipolar or multipolar body cells,and most of cells were died after 48 hours.The 40%-50% neuronspecific enolase,35%-40% glial fibrillary acidic protein was found to be positive.The positive rate of nidogen was temporary increased to 63% at hour 2 after culture；however,it was decreased to 1.6% after 48 hours.CONCLUSION:BMSCs can differentiate into neuronal-like cells,as well as inhibit lymphocyte proliferation in MLR system,which possess down regulation effect on alloimmunity-reaction.

Objective To investigate drug resistance genes and epidemic characteristics of β-lactamase carried by carbapenem-resistant Acinetobacter baumannii (CRAB) in the respiratory intensive care unit(RICU) in a hospital.Methods Clinically isolated CRAB from RICU patients in October-December 2015 were collected.Five drug resistance genes (KPC-2,IMP,VIM,NDM-1,OXA-23) were specifically amplified by polymerase chain reaction (PCR),amplified products were performed agarose gel electrophoresis and sequencing analysis,the homology was analyzed with pulsed-field gel electrophoresis (PFGE).Results A total of 22 CRAB strains were isolated in October-December 2015,19 (86.36％) of which were isolated from sputum.The resistance rate of 22 CRAB strains to compound sulfamethoxazole was 59.09 ％,resistance rate to minocycline was 9.09 ％,all were sensitive to polymyxin B,resistance rates to other antimicrobial agents were more than 80％.Three kinds of resistance genes KPC-2,IMP and NDM-1 were not found by PCR amplification,positive rates of VIM and OXA-23 were both 100％.PFGE homology analysis revealed that 22 strains were divided into 13 different types,each type contained 1-5 strains,9 types(69.23％) contained only 1 strain respectively,the other 4 types (30.77％) contained 2-5 strains.A5,A7,and A8;A9,A11,A14,A19 and A22;A4,A10 and A12;A16 and A18 were of the same type respectively.Conclusion The main types of β-lactamase-resistant genes of CRAB in RICU are VIM and OXA-23.Homology analysis shows a small parts are of the same clone strains,which reveals epidemic of a small scale.