Objective To analyze the association of Crohn's disease(CD)with vitamin D receptor(VDR) gene polymorphisms. Methods After collecting 326 CD patients and 464 healthy controls,the four single nucleotide polymorphisms of VDR (FokI, BsmI, ApaI and TaqI) were examined by a SNaPshot technique. Results Compared with those in controls,the frequencies of mutant allele(A)and genotype(GA+AA)of BsmI were significantly decreased in CD patients(both P=0.001). The similar conclusions were also drawn for the mutant allele(C)and genotype(TC+CC)of TaqI(both P<0.05). In further stratified analysis,compared with those in controls,the mutant alleles and genotypes of BsmI and TaqI were significantly reduced in stenotic type CD patients (all P<0.0083). The analyses of linkage disequilibrium(LD)and haplotype showed that BsmI,ApaI and TaqI were in a strong LD,and the formed haplotype AAC was significantly lower in CD patients than that in controls (P <0.05). Conclusions VDR(BsmI and TaqI)polymorphisms are significantly related with the reduced susceptibility to CD,especially for patients with stenotic CD. Moreover,the haplotype AAC might engender a reduced risk of CD.

Objective To investigate the association of ulcerative colitis (UC) with fork head/ winged helix transcription factor-3 (Foxp3) polymorphisms in Han population in Zhejiang province,China.Methods A total of 381 UC patients and 490 healthy controls were enrolled in this study.The four single nucleotide polymorphisms (SNPs) of Foxp3 (rs3761547,rs2232365,rs2294021,rs3761548) were examined by SNaPshot.The analyses of linkage disequilibrium (LD) and haplotype were also performed in all study subjects.Results When male and female UC patients were compared with their corresponding controls respectively,the alleles and genotypes of the four SNPs were not statistically different (all P ＞0.05).According to severity and location of the disease,the UC patients were divided into different subgroups.The alleles (C,G,A) of (rs2232365,rs2294021,rs3761548) were more frequent in male patients with severe UC than in the male controls (69.6％ vs 34.3％,P =0.001;69.6％ vs 34.3％,P =0.001;39.1％ vs 14.4％,P =0.002,respectively).As compared with the female controls,the alleles (C,G,A) and genotypes (TC + CC,AG + GG,CA + AA) of (rs2232365,rs2294021,rs3761548) were significantly increased in the female patients with severe UC (51.9％ vs 38.0％,63.5％ vs 39.2％,53.8％ vs21.4％,80.8％ vs57.7％,84.6％ vs58.4％,76.9％ vs34.7％,all P＜0.05).The four SNPs above were shown to be in a strong LD both in male and in female subjects.When male and female UC patients were compared with their corresponding controls respectively,nevertheless,each haplotype frequency was not statistically different (all P ＞ 0.05).Conclusions Foxp3 (rs2232365,rs2294021,rs3761548) variations might engender the increased risk of severe UC in Chinese Han patients.

Objective To explore the association of Crohn's disease (CD) with T cell immunoglobulin and mucin domain 3 (Tim-3) gene polymorphisms in patients of Zhejiang Han population in China.Methods A total of 308 CD patients and 573 age-and sex-matched healthy controls were enrolled in our study.Two single nucleotide polymorphisms (SNPs) of Tim-3 (rs1036199 and rs10515746) were examined by the improved multiple ligase detection reaction technique (iMLDR).Analyses of linkage disequilibrium and haplotype were also performed by Haploview 4.2 software in all study subjects.Results In general,the allele and genotype frequencies of Tim-3 (rs1036199 and rs10515746) were not statistically different between CD patients and the controls (all P ＞0.05).According to the Montreal Classification,CD patients were divided into different subgroups.The variant allele (C) and genotype (AC + CC) of rs1036199 were more frequent in CD patients with penetrating diseases than in the controls (10.4％ vs 1.7％,P =0.002;20.8％ vs 3.5％,P =0.023).Similar conclusions were also drawn for the variant allele (A) and genotype (CA + AA) of rs10515746 in patients with penetrating diseases when compared with the controls (10.4％ vs 2.2％,P =0.000;20.8％ vs 4.2％,P =0.033,respectively).The two SNPs of Tim-3 were in strong linkage disequilibrium (D'=1.0,r2 =0.928).The haplotype (AC) formed by their wild-type alleles (A) and (C) was decreased in patients with penetrating CD compared with the controls (89.6％ vs 98.3％,P =0.000).However,the haplotype (CA) formed by their variant alleles was more frequent in patients with penetrating CD than in the controls (10.4％ vs 1.6％,P =0.000).Conclusions Tim-3 (rs1036199 and rs10515746) variations might be correlated with the enhanced risk of penetrating diseases in CD patients.Furthermore,the haplotype (AC) and (CA) formed by the two SNPs might be a protective and a risky factor for penetrating CD respectively.

AIM:To explore the protective effects of exosome secreted by human umbilical mesenchymal stem cells on cardiac fibrosis in diabetic mouse model.METHODS:Male C57BL/6 mice at 6～8 weeks of age were divided in-to 3 groups randomly:control group ,diabetes mellitus(DM)group and DM+exosome group.To develop mouse DM mo-del,the mice were fed with high-fat diet for 5 weeks,followed by intraperitoneal injection of 45 mg/kg streptozocin once a week for 5 weeks.It was considered as a successful DM model that the blood glucose of the mice was ≥16.7 mmol/L.The mice in DM+exosome group were injected with exosome via tail vein.The mice in other 2 groups were injected with saline at the same volume.The heart function was evaluated by color Doppler echocardiography for small animals.The blood sam-ples were collected from abdominal aortas.The blood glucose and non-esterified fatty acids were measured by biochemical colorimetric assay.HE staining was performed to observe the structural changes of myocardial fibers ,and Masson staining was used to observe the cardiac fibrosis.RESULTS:The results of echocardiography showed that left ventricular end-dias-tolic dimension(LVIDd)and left ventricular end-systolic dimension(LVIDs)of diabetic mice were larger than those incontrol mice(P<0.05 and P<0.01,respectively).The ejection fraction(EF)and fractional shortening(FS)decreased in the diabetic mice(P<0.01).Exosome treatment significant decreased the LVIDs(P<0.01),but increased the EF and FS(P<0.01).The blood glucose and non-esterified fatty acids were significantly increased in the diabetic mice.The injection of the stem cell exosome significantly decreased the blood glucose and non -esterified fatty acids(P<0.01).HE staining observation showed that cardiomyocyte hypertrophy and fragmentation of cardiomyocyte in DM group were more se -rious than those in control group.Masson staining showed that the area of fibrosis in DM group was larger than that in con-trol group(P<0.01),but that in DM+exosome group was reduced(P<0.01).CONCLUSION:Exosome secreted by human umbilical mesenchymal stem cells protects the DM model mice from cardiac fibrosis.

This study purposed to investigate the effects of different oxygen concentrations and reactive oxygen species (ROS) on the biological characteristics of hematopoietic stem cells (HSC) and their possible mechanisms through simulating oxygen environment to which the peripheral blood HSC are subjected in peripheral blood HSCT. The proliferation ability, cell cycle, directed differentiation ability, ROS level and hematopoietic reconstitution ability of Lin(-)c-kit(+)Sca-1(+) BMHSC were detected by using in vitro amplification test, directional differentiation test, cell cycle analysis, ROS assay and transplantation of Lin(-)c-kit(+)Sca-1(+) HSC from sublethally irradiated mice respectively. The results showed that oxygen concentrations lower than normal oxygen concentration, especially in hypoxic oxygen environment, could reduce ROS generation and amplify more primitive CD34(+)AC133(+) HSC and active CD34(+) HSC, and maintain more stem cells in the G(0)/G(1) phase, which is more helpful to the growth of CFU-S and viability of mice. At the same time, BMHSC exposed to normal oxygen level or inconstant and greatly changed oxygen concentrations could produce a high level of ROS, and the above-mentioned features and functional indicators are relatively low. It is concluded that ROS levels of HSC in BMHSCT are closely related with the oxygen concentration surrounding the cells and its stability. Low oxygen concentration and antioxidant intervention are helpful to transplantation of BMHSC.
Animals ; Bone Marrow Cells ; cytology ; metabolism ; Cell Differentiation ; Cells, Cultured ; Female ; Hematopoietic Stem Cells ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Oxygen ; administration & dosage ; pharmacology ; Reactive Oxygen Species ; metabolism

To investigate the effect of recombinant human growth hormone (rhGH) on JAK2-STAT3 pathway and the growth of gastric cancer cell lines at different GHR expression status, the eukaryotic expression vector targeting human GHR (pGPU6/GFP/Neo-shGHR and pGPU6/GFP/Neo-scramble) was constructed and transfected into MGC803 cells by Lipofectamine 2000. Stable expressive cell lines were obtained by G418 screening. The expression of GHR was analyzed by Western blotting. After being stimulated with rhGH, cell growth was detected by MTT assay. Cell cycle and apoptosis were examined by flow cytometry. The components of JAK2/STAT3 signaling pathway were detected by Western blotting. There is no significant difference of GHR expression between MGC803 and pGPU6/GFP/Neo-scramble-transfected cells (named as MGC803-NC) (P > 0.05). Compared with MGC803, the GHR expression in pGPU6/GFP/Neo-shGHR-transfected cells (named as MGC803-shGHR) decreased significantly (protein decreased 50%). The cells were treated with rhGH at 0, 150 and 300 ng x mL(-1), the growth rate of MGC803 and MGC803-NC increased significantly, PI and the number of G2/M phase cells all increased significantly, and apoptosis decreased significantly. Western blotting revealed that the expression of pJAK2 and pSTAT3 was up-regulated after being treated with rhGH in MGC803 and MGC803-NC cells. In contrast, similar change was not observed in MGC803-shGHR cells. Knockdown of GHR gene may decrease the sensitivity of gastric cancer cells to rhGH, and down-regulating of components of the expression of JAK2/STAT3 signaling pathway may be the potential mechanisms.
Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Human Growth Hormone ; genetics ; pharmacology ; Humans ; Janus Kinase 2 ; metabolism ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptors, Somatotropin ; genetics ; metabolism ; Recombinant Proteins ; genetics ; pharmacology ; STAT3 Transcription Factor ; metabolism ; Signal Transduction ; Stomach Neoplasms ; metabolism ; pathology ; Transfection

OBJECTIVETo establish a gas chromatographic method for determination of halogenated alkanes and aromatic hydrocarbons including trichloromethane, 1,2-dichloroethane, carbon tetrachloride, benzene, toluene, ethylbenzene and xylene in the air of workplace.

METHODSAfter the air samples collected with activated carbon tubes and desorbed with CS(2), the target toxicants were separated with FFAP capillary columns and detected with flame ionization detector.

RESULTSThe coefficient of correlation was above 0.999 and the lowest detectable concentrations were 0.2 ∼ 3.6 mg/m(3) with the RSD of 1.2% ∼ 4.6%. The desorption efficiencies was 94.9% ∼ 100.7%.

CONCLUSIONThe method shows lower detection limit, high accuracy and precision. It is feasible for determination of the seven halogenated alkanes and aromatic hydrocarbons in the air of workplace.

Atrioventricular Block ; etiology ; Cardiac Catheterization ; adverse effects ; Heart Septal Defects, Ventricular ; therapy ; Hematologic Diseases ; etiology ; Hemolysis ; Humans

Objective:To investigate the association between metabolically healthy obesity(MHO) and atherosclerosis risk among Chinese community population aged 40 or older.Methods:A total of 9 525 participants without cardiovascular diseases (3 621 men and 5 904 women) from Jiading community in Shanghai were enrolled to complete questionnaires, undergo extensive physical examination including brachial-ankle pulse wave velocity (baPWV) and blood pressure (BP) assessment, and laboratory screening. According to body mass index (BMI) and metabolic status, these participants were categorized into 4 groups including metabolically healthy non-obese (MHNO), metabolically unhealthy non-obese (MUNO), MHO, and metabolically unhealthy obese (MUO). High baPWV was defined as baPWV>1 400 mm/s, and high pulse pressure (PP) was defined as PP above fourth quartile of the population. Multivariate logistic regression model was conducted to explore the relationship between MHO and high baPWV as well as high PP after adjusting for confounders. Results:After multivariable adjustment, such as sex, age, current smoking, current drinking, and education, logistic regression analysis showed that MHO was significantly correlated with high baPWV ( OR=1.18, 95% CI 1.02-1.37) and high PP ( OR=1.72, 95% CI 1.43-2.08) in comparison with MHNO. Otherwise, both MUNO and MUO subjects were at higher risk for suffering from high baPWV (MUNO: OR=3.02, 95% CI 2.60-3.50; MUO: OR=3.26, 95% CI 2.87-3.70) and high PP (MUNO: OR=2.56, 95% CI 2.17-3.02; MUO: OR=3.49, 95% CI 3.01-4.06). Conclusion:On the basis of Chinese community population, there was a pronounced correlation between the MHO phenotype and the increased risk of developing atherosclerosis.

Classical swine fever virus (CSFV), an enveloped positive-stranded RNA virus in the genus Pestivirus of the Flaviviridae family, is the causative agent of a highly contagious swine disease characterized by symptoms of hemorrhagic fever and immune depression, usually leading to substantial economic losses. The serological methods for detection of CSFV antibody such as ELISA are important means for the diagnosis of CSFV and immune surveillance. It is difficult to obtain CSFV antigen with high quality using traditional method because its titration titer is low in cell culture. CSFV has four structural protein named C, E0, El and E2. The E2 protein contains major antigenic determinants that are conserved between different CSFV strains and involved in neutralization by antibodies. So recombinant E2 protein can be developed as an alternative to the intact viral antigen. So far, CSFV E2 have not been expressed in E. coli with high level. Many factors, such as the secondary structure, the stability of 5' and 3' terminus of gene, the location of SD sequence and the bias of codes, are involved in the expressing level of foreign gene in E. coli . In this study, two sites of the E2 gene sequence were confirmed to be detrimental to its expression efficiency in E. coli through the computer-aided analysis. So they were mutated using recombinant PCR without changing the amino acids sequence of CSFV E2 gene. A plasmid was constructed by inserting the mutated E2 gene into the prokaryotic expression vector pET-28a(+) and named pETE2. The E. coli competent host BL21 (DE3)lysS transformed with pETE2 could express the E2 gene at high level, amounting to 28% of the total protein of the induced recombinant bacteria at the presence of IPTG. Except the hydrophobic transmembrane domain at C terminus, the recombinant E2 protein includes the total aa sequence. So it contains all the potential linear antigen epitopes of E2 protein because hydrophobic aa region can not form epitope. The recombinant E2 protein was CSFV-specific as proved by Western blotting and indirect ELISA. The rabbits immunized with the recombinant E2 can be protect from the challenge of hog cholera lapinized virus. This is the first report that E2 gene is expressed with high level expression in E. coli. In conclusion, it is an effective measure that mutate the CSFV E2 gene to increase its expression level in E. coli. The recombinant CSFV E2 protein possess fine immunonicity and can be used the antigen for the detection of CSFV antibody.
Antigens, Viral ; genetics ; immunology ; metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Mutagenesis, Site-Directed ; methods ; Polymerase Chain Reaction ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Envelope Proteins ; genetics ; immunology ; metabolism