OBJECTIVETo observe the differentiation of bone mesenchymal stem cells (BMSCs) co-cultured with purified sinoatrial node cells (SNC) of neonate rats.

METHODSSNC from neonatal SD rat were cultured and purified with differential attachment method and labeled with BrdU. Rat BMSCs were isolated by a Percoll's gradient solution and cultured in DMEM. After 2 passages, these BMSCs were transfected with pEGFP-N1 by Lipofectamine and labeled with GFP. EGFP-BMSC were co-cultured with SNC in a rate of 1:5 for 1 week. EGFP-BMSC cultured in SNC culture medium served as controls. SNC marker hyperpolarization activated cyclic nucleotide gated cation channel 4 (HCN4) and connexin 45 (Cx45) expressions were determined by immunofluorescence staining.

RESULTPositive immunofluorescence staining against HCN4 and Cx45 were detected in EGFP-BMSC co-cultured with SNC but not in EGFP-BMSC cultured in SNC culture medium.

CONCLUSIONDirect cell-to-cell contact between BMSCs and SNC cells may induce BMSCs differentiation into sinus node-like cells.

Animals ; Bone Marrow Cells ; cytology ; Cell Culture Techniques ; Cell Differentiation ; Cell Separation ; Cells, Cultured ; Coculture Techniques ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley ; Sinoatrial Node ; cytology

OBJECTIVESTo explore serum cytokines levels (including IL-1beta, sIL-2R, IL-6, TNF-alpha, and IFN-upsilon) and their significance in patients with acute coronary syndrome (ACS) and the subsequent follow-ups, with attempt to estimate the role of various serum inflammatory markers in the diagnosis and assessment of ACS.

METHODSThe study population include 40 patients with acute myocardial infarction (AMI), 40 patients with unstable angina pectoris (UAP), and 40 controls. Among the 80 patients, 60 patients attended a follow up 4 months later. Serum inflammatory markers including IL-1beta, slL-2R, IL-6, TNF-alpha, and IFN-upsilon were measured by enzyme linked immunosorbent assay.

RESULTSSerum IL-1beta, sIL-2R, IL-6, TNF-alpha were significantly higher in AMI group or UAP group compared to the control group and became significantly lower 4 months later in the follow-up patients. Serum levels of IFN-upsilon shows no significant difference between AMI group or UAP group and controls, also showing no significant change when measured in follow up patients. There was no correlation between serum creatine kinase-MB isoenzyme levels and serum inflammatory markers either in UAP or AMI group. Furthermore, when divided into two subgroups using Wagner's QRS scoring system in the AMI group, there is no difference of each serum inflammatory marker between < or = 6 scores group and > 6 scores group.

CONCLUSIONSerum levels of certain inflammatory markers may have some diagnostic value for ACS, and can be a useful marker reflecting disease stability.

Aged ; Angina, Unstable ; blood ; diagnosis ; Biomarkers ; blood ; Creatine Kinase ; blood ; Creatine Kinase, MB Form ; Cytokines ; blood ; Female ; Follow-Up Studies ; Humans ; Interferon-gamma ; blood ; Interleukin-1 ; blood ; Interleukin-6 ; blood ; Isoenzymes ; blood ; Male ; Middle Aged ; Myocardial Infarction ; blood ; diagnosis ; Receptors, Interleukin-2 ; blood ; Tumor Necrosis Factor-alpha ; metabolism

AIMTo determine the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in the expression of endothelin receptor type B (ETB) during culture.

METHODSSB386023, a specific inhibitor for ERK1/2 pathway, was used to define the intracellular signaling pathway for the upregulation of ETB receptors and sarafotoxin 6c (S6c), a selective agonist for ETB receptors, induced contraction in isolated rat superior mesenteric arteries. The contraction was recorded by a sensitive in vitro myograph and the receptor mRNA was quantified by a real-time PCR. The phosphorylated ERK1/2 proteins were analyzed by phosphoELISA assay.

RESULTSS6c induced strong contractile responses of the artery after culture for 24 h, while there was no response to S6c in fresh vessel segments. The enhanced contractile response to S6c paralleled with an increase of mRNA for ETB receptors. The phosphorylated ERK1/2 proteins significantly increased after culture for 3 h. After co-culture with SB386023 for 24 h, S6c-induced contractions significantly decreased with reduction of Emax from (217 +/- 14) % to (127 +/- 23) % (P <0.01). This response paralleled with a decreased level of ETB receptor mRNA.

CONCLUSIONERK1/2 pathway was involved in the up-regulation of ETB receptors on smooth muscle cells isolated from rat mesenteric arteries during culture.

Animals ; Cells, Cultured ; Male ; Mesenteric Arteries ; cytology ; Mitogen-Activated Protein Kinase 1 ; antagonists & inhibitors ; metabolism ; Mitogen-Activated Protein Kinase 3 ; antagonists & inhibitors ; metabolism ; Muscle Contraction ; drug effects ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Organ Culture Techniques ; Phosphorylation ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin B ; biosynthesis ; genetics ; Signal Transduction ; Up-Regulation ; Vasoconstrictor Agents ; pharmacology ; Viper Venoms ; pharmacology

OBJECTIVE: To detect the levels of index related to inflammation such as soluble CD40 ligand (sCD40L), neutrophil collagenase-8 (MMP-8), and pregnancy associated plasma protein-A (PAPP-A) , lipid peroxidation and autoimmune indexes such as oxidized low density lipoprotein (ox-LDL) and its antibody (ox-LDL Ab) in patients with coronary heart disease, and to investigate its relationship with acute coronary syndrome (ACS). METHODS: Contents of sCD40L, MMP-8, PAPP-A, ox-LDL and ox-LDL Ab in the peripheral blood were measured by enzyme-linked immunosorbent assay from 109 patients with coronary heart disease including 36 acute myocardial infarction (AMI), 38 unstable angina pectoris (UAP), and 35 stable angina pectoris (SAP) and 36 controls without coronary heart disease. RESULTS: The levels of each index in the peripheral blood of ACS patients (including AMI and UAP) were higher than those of SAP patients and controls (P < 0.05), and the difference of each index between UAP group and AMI group in ACS patients had no statistical significance (P > 0.05). The levels of each index of SAP patients, except PAPP-A, were all higher than those of controls (P <0.05). All the indexes were helpful in diagnosis of ACS. The area under the ROC curve of each index is between 0.7 and 0.9. CONCLUSION The increase of sCD40L, MMP-8, PAPP-A, ox-LDL and ox-LDL Ab levels in peripheral blood may be related to the pathogenesis of ACS, and can be used as potential markers of unstable atherosclerosis plaque.
Aged ; Angina, Unstable ; blood ; immunology ; Autoantibodies ; blood ; Biomarkers ; blood ; CD40 Ligand ; blood ; Coronary Artery Disease ; blood ; immunology ; Female ; Humans ; Lipoproteins, LDL ; blood ; immunology ; Male ; Matrix Metalloproteinase 8 ; blood ; Middle Aged ; Myocardial Infarction ; blood ; immunology ; Pregnancy-Associated Plasma Protein-A ; metabolism

OBJECTIVETo construct the sodium channel gene SCN5A-delQKP1507-1509 mutation associated with congenital long QT syndrome, and its eukaryotic expression vector, and to examine the expression of mutation protein in human embryonic kidney (HEK) 293 cells.

METHODSEukaryotic expression vector PEGFP-delQKP-hH1 for SCN5A-delQKP1507-1509 mutation was constructed by rapid site-directed mutagenesis. HEK293 cells were transfected with the wild or mutant vector using lipofectamine, and then subjected to confocal microscopy. The transfected cells were immunostained to visualize intracellular expression of the mutant molecules.

RESULTSDirect sequence and electrophoresis analysis revealed 9 basic group absences at position 1507-1509. The delQKP1507-1509 mutation eukaryotic expression vector was expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both wild type and mutant molecules on the plasma membrane and there was no difference in the amount of protein, which suggested that the mutant delQKP1507-1509 did not impair normal protein expression in HEK293 cells.

CONCLUSIONSSuccessful construction of mutant SCN5AdelQKP1507-1509 eukaryotic expression vector and expression of SCN5A protein in HEK293 cells provides a basis for further study on the functional effects of congenital long QT syndrome as a cause of SCN5A mutation.

Blotting, Western ; HEK293 Cells ; Humans ; Long QT Syndrome ; congenital ; genetics ; Mutagenesis, Site-Directed ; NAV1.5 Voltage-Gated Sodium Channel ; analysis ; genetics ; physiology

OBJECTIVETo investigate the changes in circulating bone marrow stem cells in pregnant rabbits after AMI (AMI) and their relationship with estradiol.

METHODSThree groups of rabbits were used, namely pregnancy and AMI group, AMI group without pregnancy, and sham operation group with pregnancy. The ratio of CD90(+) cells in the peripheral blood was determined with flow cytometry in all the rabbits, and serum estradiol level measured. Four weeks after AMI, hemodynamic measurements were carried out. The morphological changes of the myocardial tissues were examined with ImageJ 1.31.

RESULTS AND CONCLUSIONFour weeks after AMI, the two pregnancy groups showed a higher Left ventricular end systolic pressure(LVESP) and+dp/dtmax, lower left ventricular end-diastolic pressure (LVEDP) and -dp/dtmax and high levels of CD90(+) cells in peripheral blood than AMI group without pregnancy (P<0.01). The ratio of circulating CD90(+) cells increased gradually with gestational age and peaked at the end stage of pregnancy. After delivery the circulating CD90+ cell ratio decreased sharply, showing a significant correlation with serum estradiol level (r=0.725, P<0.01). Four weeks after AMI, the pregnancy group had smaller myocardial infarction (MI) volume than the non-pregnant group (22.17+/-6.34% vs 38.86+/-5.97%, P<0.05). Circulating bone marrow stem cells increased during pregnancy with gestational age and peaked at the end stage of pregnancy. Ten days after delivery, the stem cells resumed basically the normal level. The proportion of circulating bone marrow stem cells was significantly correlated with the level of serum estradiol during pregnancy, and mobilization of the bone marrow stem cells induced by acute ischemic event in pregnant rabbits was advanced. 4 weeks after AMI, the pregnant rabbits showed better heart contraction and diastolic function than the non-pregnant ones.

Animals ; Bone Marrow Cells ; cytology ; Estradiol ; blood ; Female ; Hematopoietic Stem Cells ; cytology ; Myocardial Contraction ; Myocardial Infarction ; blood ; physiopathology ; Pregnancy ; Pregnancy Complications, Cardiovascular ; blood ; Rabbits ; Thy-1 Antigens ; blood ; Time Factors

OBJECTIVESTo understand the relation between nitric oxide (NO) level in seminal plasma and male fertility.

METHODSThe levels of NO in seminal plasma of 174 fertile males and 217 abnormospermia patients were measured by using the reductase of nitric acid, Greiss reagent and spectrophotometry.

RESULTS1. NO was found in all 174 samples (20-49 years) of fertile males which was (27.78 +/- 5.81) mumol/L. The NO level in seminal plasma in fertile males was became higher after age 40, and there was no significant difference between 20-29-year-old [(26.25 +/- 5.52) mumol/L] and 30-39-year-old[(28.11 +/- 5.87) mumol/L]. But the group of 40-49-year-old[(30.17 +/- 6.14) mumol/L] had a higher level of NO in seminal plasma than 20-29-year-old (P < 0.05). 2. The seminal plasma samples from nine types of abnormospermia were measured, which all had a higher level of NO than fertile males. In abnormospermia, the level of NO in seminal plasma of the patients with single abnormality increased slightly, two abnormality obviously increased, and the highest level of NO in seminal plasma appeared in three abnormality.

CONCLUSIONSThis results confirmed that proper level of NO in seminal plasma may regulate the spermatogenesis.

Adult ; Case-Control Studies ; Humans ; Infertility, Male ; metabolism ; Male ; Middle Aged ; Nitric Oxide ; metabolism ; Semen ; metabolism ; Spectrophotometry

OBJECTIVETo investigate the changes in plasma levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) and in peripheral CD34(+) cells in patients with acute myocardial infarction (AMI), and explore their role in AMI.

METHODSEnzyme-linked immunoassay (ELISA) was employed for measuring the levels of VEGF and SDF-1 in AMI patients on days 1, 3, 7, 10, and 14 of onset and in normal control subjects. The absolute counts of CD34(+) in the peripheral blood were measured on days 1, 7, and 14 by flow cytometry in AMI patients, with their myocardial enzyme and troponin I detected and electrocardiography (ECG) and echocardiography (UCG) recorded.

RESULTSPeripheral CD34(+) cells obviously increased on day 7 after AMI onset (2.35-/+0.72/microl vs 1.48-/+0.49/micro, P<0.05). VEGF levels were significantly higher in AMI patients than in the control subjects, reaching the peak level and on day 14 (197.56-/+39.87 vs 53.79-/+18.12 pg/ml, P<0.01). SDF-1 level obviously decreased on day 1 after AMI onset (1683.12-/+224.79 vs 2178.67-/+265.34 pg/ml, P<0.01), followed by gradually increased to the control level. Obvious correlation was noted between the level of VEGF on day 7 and the peak level of peripheral CD34(+) cells, and the peak plasma VEGF level was obviously associated with the peak serum CK-MB and troponin I levels.

CONCLUSIONThe stem cells are mobilized into the peripheral blood in the event of AMI. Obviously increased VEGF level following AMI may persist for at least 2 weeks, whereas SDF-1 level undergoes temporary decrement after AMI. The dynamic changes of VEGF and SDF-1 can be related to the mobilization and homing of the stem cells to the injured myocardium.

Adult ; Antigens, CD34 ; blood ; Chemokine CXCL12 ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Humans ; Male ; Myocardial Infarction ; blood ; Time Factors ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo evaluate the effect of chronic virus infection on laboratory tests results in patients with osteoarticular tuberculosis.

METHODSA total of 121 patients with osteoarticular tuberculosis, who were hospitalized in Shenzhen Third People's Hospital during June 2008 to June 2012, were recruited for analysis. Clinical laboratory tests results were collected for comparison between patients with or without chronic co-infection with virus.

RESULTSAmong the 121 patients, thirty patients were co-infected with hepatitis B virus (HBV), two were with Human immunodeficiency virus (HIV), and one was co-infected with HBV, HIV and hepatitis C virus (HCV). Compared to patients with osteoarticular tuberculosis without HBV/HCV/HIV infection, patients with chronic HBV/HCV/HIV virus infection had similar positive rate of laboratory tests including tissue smear acid-fast bacilli (AFB) staining, tissue Mycobacterium tuberculosis (Mtb) culture, tissue Mtb DNA detection, serological test of antibodies against Mtb, and Mtb. antigen-specific interferon-gamma release assay. Similar results were also found for erythrocyte sedimentation rate, C-reative protein level and liver function including Alanine aminotransferase and Aspartate Aminotransferase.

CONCLUSIONChronic infection with HBV/HCV in patients with have no obvious effect on clinical laboratory tests related to tuberculosis.

Adult ; Female ; HIV ; genetics ; isolation & purification ; physiology ; HIV Infections ; complications ; virology ; Hepacivirus ; genetics ; isolation & purification ; physiology ; Hepatitis B virus ; genetics ; isolation & purification ; physiology ; Hepatitis B, Chronic ; complications ; virology ; Hepatitis C ; complications ; virology ; Humans ; Male ; Middle Aged ; Mycobacterium tuberculosis ; genetics ; isolation & purification ; physiology ; Tuberculosis, Osteoarticular ; etiology ; microbiology ; virology

OBJECTIVE: To investigate the role of adult cardiomyocytes in the differentiation of mesenchymal stem cells (MSCs) into cardiomyocytes. METHODS: Rat MSCs were isolated by a Percoll's gradient solution and cultured in low-glucose Dulbecco' s modified Eagle' s medium (DMEM). After 2 passages, cell-surface antigen CD34, CD71 and CD90 for rat MSCs were determined by flow cytometry, and these MSCs were transfected with pEGFP-N3 by Lipofectamine2000. Then those MSCs labeled with GFP, were cultured in contacted, nocontacted and conditioned with adult rat myocardiocytes. Immunofluorescence staining against alpha-actin, desmin, and troponin-T were performed after 1 week. RESULTS: Immunofluorescence staining was positive against alpha-actin, desmin, and troponin-T on MSCs in contacted culture group. In contrast, no alpha-actin, desmin, and troponin-T expression on MSCs were observed in the noncontacted culture group and the conditioned culture group. CONCLUSION Direct cell-to-cell contact between MSCs and adult cardiomyocytes may induce differentiation of MSCs into cardiomyocytes.
Animals ; Antigens, CD34 ; analysis ; Bone Marrow Cells ; cytology ; Cell Communication ; Cell Differentiation ; physiology ; Cell Separation ; Cells, Cultured ; Coculture Techniques ; Female ; Male ; Mesenchymal Stem Cells ; cytology ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Sprague-Dawley ; Thy-1 Antigens ; analysis