0.05).Conclusions The expression of myostatin gene mRNA is increased in myotonic dystrophy.Up-regulated expression of myostatin in skeletal muscle might be associated with the mechanism of myotonic dystrophy.

Objective To discuss the application value of modified Hodge test(MHT) for screening carbapenemase-producing Enterobacteriaceae.Methods The 24 Enterobacteriaceae reduced susceptibility to carbapenems were detected by MHT.At the same time,polymerase chain reaction(PCR) was used to detect carbapenemase genes of KPC,NDM,IMP,SIM and VIM.PCR products were sequenced and the results were compared with the sequences of Gen Bank database.Comprehensive analysis the application value of MHT and PCR to detect carbapenemase.Results Among these 24 strains,13 stains appeared to produce carbapenemase by MHT,5 positive strains were found to carry carbapenemase genes by PCR.By comparing with the sequences of Gen Bank database 1 strain were confirmed to KPC-2 and 4 strains were confirmed to IMP-4.We found that 4 strains of Enterobacteriaceae,detected carbapenemase by MHT and PCR at the same time.9 strains of MHT were positive,but we couldn′t detect the carbapenemase genes.1 strain of MHT was negative,but carbapenemase gene was found in the strain.Conclusion The value of MHT to screen carbapenemase-producing Enterobacteriaceae is necessary to further study.

To study the contribution of T cell subsets in the pathogenesis of Murine hepatitis virus Type3 (MHV-3) induced chronic viral hepatitis in C3H/Hej mice, ninety C3H/Hej mice were chosen to individually receive 10 plaque forming units (PFU) of MHV-3 intraperitoneally. The changes of virus titer and pathology in liver tissue were examined by standard plaque assay and by the hematoxylin/eosin (HE) staining method from 2 days post MHV-3 infection. The ratios of T cell subsets including CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4-CD8-, CD3+CD4+CD25+, CD3+CD4+CD25- and CD3+CD4-CD25+ T lymphocyte of total T lymphocytes in blood, spleen and liver were examined at 0, 2, 4, 6,8, 10, 12, 15, 20, 25, 30, 40 days post MHV-3 infection by flow cytosorting. We observed that the virus titer raised and showed persistent virus duplications and inflammatory changes in the livers of C3H/Hej mice from 2 days post MHV-3 infection. The double negative T cell (DN Treg cell) and CD4+CD25+ T cell ratios increased significantly from 2 days post MHV-3 infection in C3H/Hej mice, and CD3+CD4+CD8-, CD3+CD4-CD8+, CD3+CD4+CD25- and CD3+CD4-CD25+ T cell ratios decreased accordingly. In conclusion, the changes of virus titer and pathology in the livers of C3H/Hej mice post MHV-3 suggest their contribution to viral persistence. Further characterizations of DN Treg cells are that infection indicates that MHV-3 could induce the chronic inflammation in livers of C3H/Hej mice.The increase of the DN Treg cell and CD4+CD25+ T cell ratios in C3H/Hej mice post MHV-3 infection suggests that DN Treg cells and CD4+CD25+ T cells may both have important suppressive immunomodulation functions in the development of chronic viral hepatitis and have important roles in the virus persistent infection. Further characterizations of DNT cell and CD4+CD25+ T cell are under investigation.

This study was designed to explore the RNA interference technique in inhibition of the expression of the mouse fibrinogen like protein 2 (mfgl2), which has been reported to be involved in the development a variety of diseases including fulminant viral hepatitis. A plasmid named p-mfgl2shRNA,complementary to the sequence of mfgl2 was constructed, while another short hairpin RNA (shRNA)which was a mutated form of the mfgl2shRNA sequences was used as a control. A plasmid named pEGFP-mfgl2 expressing the mfgl2-EGFP fusion protein was also constructed for the screening of the effect of p-mfgl2shRNA on mfgl2 expression. By cotransfection of p-mfgl2shRNA and pEGFP-mfgl2 or pcDNA3.1-mfgl2 expression construct into CHO cells or HeLa cells, the inhibition of mfgl2 expression by mfgl2shRNA was analyzed by direct observation through fluorescent microscopy, FACS, RT-PCR and immunohistochemistry staining. The experiments showed the significant inhibitory effect of p-mfgl2shRNA on mfgl2 expression at 48h post-transfection in both CHO and Hela cell lines with the inhibitory efficiency as high as 80.1%. The study demonstrated that the construct of p-mfgl2shRNA successfully interfered with the mfgl2 expression in vitro.

Objective - To analyze the relationship between cobalt level of post shift urine and individual exposure level of ， cobalt and its compounds in cobalt exposed workers and to explore the feasibility of using urine cobalt as a biomarker. Methods - A total of 148 occupational cobalt exposed workers from a new material company were selected as the exposed ， - - group and 44 non occupational cobalt exposed workers from the company were selected as the control group using the typical sampling method. The exposure concentration of time weighted average of cobalt and its compounds in the workplace air of the - two groups was determined by inductively coupled plasma mass spectrometry as the individual exposure level. The cobalt levels - - of pre shift and post shift urinary samples of the two groups were detected by this method. The linear relationship between the - cobalt level of post shift urine and the individual exposure level of cobalt and its compounds in the air of the workplace was Results - 3 analyzed. The individual exposure level of cobalt and its compounds in the exposed group was 1.10 131.71 μg/m with （M） 3 the median of 12.23 μg/m. No cobalt and its compounds were detected in the workplace air in the control group. The cobalt - - levels of pre shift and post shift urines in exposed group were higher than those in the control group at the same time point （M： vs ， vs ， P ） - - 1.54 0.56 μg/L 8.77 0.83 μg/L all <0.01 . The cobalt level of post shift urine was higher than that in pre shift （M： vs ，P ）， urine in the exposed group 8.77 1.54 μg/L <0.01 and it was positively correlated with the individual exposure level （ ，P ） ， of cobalt and its compounds Spearman correlation coefficient=0.86 <0.01 . After common logarithm conversion the linear regression equation of the cobalt level of post shift urine and the common logarithm of individual exposure level of cobalt and （x） ：ŷ x（ ；F ， its compounds in the exposed group was as follows = −0.178 + 0.988 coefficient of determination=0.72 =374.75 P ；t ， P ） Conclusion - <0.01 = - 19.36 <0.01 . There was a linear correlation between cobalt level of post shift urine and occupational cobalt exposure level of cobalt exposed workers. Urinary cobalt can be used as a biomarker of occupational cobalt

Objective To investigate the relationship between apolipoprotein E (APOE) genotype and onset of hypertensive intracerebral hemorrhage (HICH). Methods One hundred and twenty-eight patients with HICH,admitted to our hospital from September 2007 to December 2010,and 84 healthy controls clinically flee from neurology disease were chosen in our study.A POE genotype was determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP); the distribution ofAPOE genotype and allele fiequency between the 2 groups were compared; the age of onset,gender,blood pressure,blood fat,bleeding part and size of haematoma in patients with different APOE genotype were further studied. Results The subjects with smoking hobby,diabetes history and higher concentrations of TC and LDL-C in the HICH group were obviously more than those in the control group (P＜0.05).As compared with those in the controls,the distributions of the APOEε3/4 and APOEε4 in the HICH group were significantly higher, and the distributions of the APOEε3/3 and APOEε3 in the HICH group were significantly lower (P＜0.05). The APOEε3/4 and APOEε4 carriers enjoyed higher percentages of onset age less than 45 years and superficial haematoma than A POEε3/3 and the A POEε3 carriers. The onset systolic blood pressure in APOEε3/4 and APOEε4 carriers was obviously lower than that in A POEε3/3 and A POEε3 carriers (P＜0.05). The plasma concentrations of LDL-C and TC in APOEε3/4 and APOEε4 carriers was significantly higher than those in APOEε3/3 and APOEε3 carriers (P＜0.05). Conclusion The APOE genotype influences the onset of HICH, and its influence factors included onset age, blood pressure,blood fat and bleeding part,which may concern with the cerebral artherosclerosis or amyloidosis.

OBJECTIVETo prepare IL-2-anchored and tumor-derived exosomes vaccine, and investigate the antitumor efficiency of the special cytotoxic T-lymphocytes induced by Ex/GPI-IL-2.

METHODSTo construct pEGFP-N1-IL2gpi plasmid coding a fusion gene of a DNA oligo encoding GPI-anchor signal sequence attaching to human IL-2 cDNA. Then T24 cell lines stably expressing GPI-IL-2 proteins (T24/GPI-IL-2) were established. Ex/GPI-IL-2 were isolated and purified by ultrafiltration and sucrose gradient centrifugation, and the morphology and molecule markers were analyzed. The mixed lymphocyte reaction study and cytotoxic study were performed to determine the proliferative effect of T lymphocytes and the cytotoxicity induced by Ex/GPI-IL-2.

RESULTSThe pEGFP-N1-IL2gpi plasmid was successfully constructed, and cell lines stably expressing GPI-IL-2 fusion proteins were established. Ex/GPI-IL-2 were small vesicular and saucer-shaped in diameter of 30-90 nm, containing heat shock protein 70, intercellular adhesion molecule-1, MAGE-1 and GPI-IL-2. Ex/GPI-IL-2-pulsed could dendritic cells induce proliferation of T cells and cytotoxic immune response more efficiently (P<0.05).

CONCLUSIONSGPI-IL-2 gene-modified tumor cells can make the exosomes containing GPI-IL-2 with an increased anti-tumor effect. Our study provides a feasible approach for exosome-based tumor immunotherapy of bladder transitional cell tumors.

Cancer Vaccines ; immunology ; Carcinoma, Transitional Cell ; immunology ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Exosomes ; genetics ; metabolism ; Glycosylphosphatidylinositols ; metabolism ; HSP70 Heat-Shock Proteins ; metabolism ; Humans ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-2 ; metabolism ; Melanoma-Specific Antigens ; metabolism ; Plasmids ; Recombinant Fusion Proteins ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; Transfection ; Urinary Bladder Neoplasms ; immunology ; metabolism ; pathology

OBJECTIVETo compare the clinical, pathological, laboratory test and follow-up data between familial and sporadic patients with distal myopathy with rimmed vacuoles (DMRV) and discuss the characteristics of this disorder in Chinese population.

METHODSThe clinical and pathological features, laboratory data and follow-up results of 33 sporadic and 4 familial cases of pathologically confirmed DMRV were summarized and compared retrospectively.

RESULTSThe patients age, onset age, or disease duration showed no significant difference between sporadic and familial cases; the onset pattern and affected muscle groups were also similar, but the sporadic cases showed more frequent dysmorphic features than the familial cases. The patients showed mild to moderate elevation of the muscle enzymes by one to three folds, and the familial patients had more significant elevation than the sporadic ones. No correlation was found between the disease duration and the level of muscle enzymes. The pathological findings were similar between the cases, and Gomori staining showed rimmed vacuoles and inclusion bodies without inflammatory cell infiltration. Follow-up results of 29 cases showed no significant difference between the two groups. The disease was slowly progressive and severely affected the quality of life of the patients, but did not produce obvious effect on the life expectancy.

CONCLUSIONThe clinical, pathological and laboratory data of Chinese DMRV patients are basically similar to those of Japanese cases. Sporadic cases tend to show more dysmorphic features than the familial ones, and occasional sporadic cases have early disease onset in early childhood.

Adult ; Asian Continental Ancestry Group ; Distal Myopathies ; classification ; genetics ; pathology ; Female ; Humans ; Inclusion Bodies ; pathology ; Male ; Pedigree ; Retrospective Studies ; Vacuoles ; pathology ; Young Adult

OBJECTIVETo investigate the biological function of platelet-derived growth factor B (PDGF-B) on the survival and proliferation of cat corneal endothelial cells so as to provide bases for further studies of its role in wound repair and its clinical application.

METHODSTotal RNA was extracted from the placenta tissues of healthy pregnant women undergoing hysterotokotomy and PDGF cDNA was obtained with reverse transcription-polymerase chain reaction (RT-PCR). The prokaryotic expression vector pET-PDGF-B was constructed and expressed the recombinant PDGF-B in Escherichia coli (E. coli) BL21 (DE3). After purification and refolding on Ni2+-chelation affinity chromatography (NTA) column, it was used to culture cat corneal endothelial cells. Cell proliferation was tested by modified tertrazolium salt (MTT) and flow cytometer. And the morphologic change and the ultrastructure were observed under an inverted phase contrast microscope, a scanning electron microscope and a transmission electon microscope, respectively.

RESULTSPDGF-B chain peptide (PDGF-BB) gene was successfully inserted into the prokaryotic expression vector, pET-28a (+). The purified recombined protein pET-PDGF-B showed a single band on sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) with the molecular weight of about 27 u, which was in agreement with the deduced value. MTT and flow cytometry showed that PDGF-BB promoted the survival and proliferation of cat corneal endothelial cells.

CONCLUSIONSThe construction of recombinant prokaryotic expression vector pET-PDGF-B and the preparation of PDGF-BB protein provide a foundation for further study of the function of PDGF-BB and producing biological PDGF-BB protein. The expressed PDGF-BB promotes the proliferation of cultured cat corneal endothelial cells.

Animals ; Cats ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cloning, Molecular ; Endothelium, Corneal ; cytology ; drug effects ; Humans ; Immunohistochemistry ; Phosphopyruvate Hydratase ; analysis ; Protein Folding ; Proto-Oncogene Proteins c-sis ; chemistry ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; isolation & purification ; pharmacology ; Wound Healing ; drug effects

OBJECTIVETo study regulation of Ca(2+)-activated K(+) channels (KCa) of mesenteric artery smooth muscle cell (SMC) from 21 old patients with essential hypertension (EH) by endothelin-1 (ET-1) and prostagl E(1) (PGE(1)).

METHODSMesenteric artery branch from EH was digested by enzyme. Patch clamp technique was used to pull cell-attached and inside-out patches on mesenteric artery SMC from EH and the normotensive patients respectively. The signal channel open probability (Po), open dwell-time (To) and close dwell-time (Tc), open channel number per patch were recorded. After adding Ca(2+) (10(-8) approximately 10(-6) mol/L), ET-1(2 approximately 8 x 10(9) mol/L) and PGE(1) (10, 20, 40, 100, 200, 400 nmol/L) to cytoplasm respectively. The parameters above were observed again.

RESULTSCompared to that of normotensive patients, the activities of KCa channels of patients with EH was higher. After adding Ca(2+) to cytoplasm,the Po of KCa channels in normotensive patients increased significantly. But it was few changes in EH group. KCa channels has dual reaction to ET-1 in normotensive patients. We have found no statistics difference when ET-1 present on KCa channels of EH cases. Whereas PGE(1) can affect KCa channels current and channels kinetic significantly in side-out patches. The Po of KCa channels increased. The To protracted and the Tc curtailed in EH.

CONCLUSIONSThe activities of KCa channels of patients with EH increased significantly. but the sensitive to Ca(2+) decreased. ET-1 were few effect to KCa channels. The PGE(1) can activated KCa channels of patients with EH.

Aged ; Alprostadil ; pharmacology ; Cells, Cultured ; Endothelin-1 ; pharmacology ; Female ; Humans ; Hypertension ; metabolism ; physiopathology ; In Vitro Techniques ; Male ; Mesenteric Arteries ; cytology ; Middle Aged ; Muscle, Smooth ; metabolism ; physiopathology ; Patch-Clamp Techniques ; Potassium Channels, Calcium-Activated ; drug effects ; metabolism