OBJECTIVETo observe the distribution and expression changes of caveolin-1 under shear stress in a turbulence flow channel model mimicking susceptible sites of atherosclerosis in vitro.

METHODSHuman umbilical vein endothelial cells (HUVECs) were inoculated to flow channel and then exposed to disturbed shear stress for 6, 12, 24 and 48 hours, respectively. HUVECs inoculated to flow channel without shear stress served as controls. Immunofluorescence staining was performed with SABC-FITC to detect the caveolin-1 distribution and RT-PCR was used to determine the mRNA expression of caveolin-1 gene in these cells.

RESULTSConfocal microcopy revealed ellipse-like shaped HUVECs under disturbed shear stress and caveolin-1 didn't move toward to the upstream edge of HUVECs but moved toward to the edge of HUVECs, and expression of caveolin-1 gene in HUVECs was significantly downregulated after exposure to shear stress for 48 hours compared to HUVECs without shear stress stimulation.

CONCLUSIONDisturbed shear stress affected disposition and expression of caveolin-1 which might be responsible for shear stress induced endothelial cell pathological atherosclerotic changes.

Caveolin 1 ; Cells, Cultured ; Endothelium, Vascular ; metabolism ; Humans ; Interleukin-8 ; Stress, Mechanical ; Umbilical Veins

OBJECTIVETo study isolation and culture from SD rat bone marrow mesenchymal stem cells (rBMMSCs) and differentiate into endothelial-like cells (ELCs) with VEGF and bFGF.

METHODSIn vitro rBMMSCs were cultured with the method of percoll (1.073 g/ml) density centrifugation and adherence to plastic dishes. Then they were seeded in LG-DMEM supplemented with 10% fetal bovine serum. The relative biologic characteristics of rBMMSCs including cell morphology, phenotype, growth curve, cell cycle and ultrastructure were studied by inverted microscope, immunocytochemistry, flow cytometry, MTT method, transmission electron microscopy(TEM). The induced cells were identified by immunocytochemistry with CD31, CD34, CD144(VE-cadherin), FITC-UEA-1 and had ability in Dil-ac-LDL uptake and were observed under inverted microscope.

RESULTSThe morphology of rBMMSCs was spindle and has whirlpool. Growth curve of P4 showed that there was difference of latency, activity and flat periods. TEM showed that there were two of rMSCs , the small of both were infantile cells. GO/G1 phase of cell cycle was 95.67% and was suggested that most of cells were not in period of proliferation. The part differentiated cells demonstrated the characters of endothelial-like cells under inverted microscope and showed the expression of CD31, CD144, CD34, and possed the functions of endothelial-like cells staining double-positive for Dil-Ac-LDL and FTIC-UEA-1.

CONCLUSION1.073 g/ml percoll density centrifugation and cultured adherence is an effective approach to obtain rBMMSCs. In vitro, the cells have potential to differentiate into endothelial-like cells

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cell Separation ; methods ; Cells, Cultured ; Endothelial Cells ; cytology ; Female ; Fibroblast Growth Factor 2 ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Vascular Endothelial Growth Factor A ; pharmacology

AIMTo explore a method to replace the isotope probe in the PCR-Select differential screening Kit with DIG labeled probe.

METHODSDifferential expressed sequences isolated from Suppression Subtractive Hybridization (SSH) was translocated onto nitric fibrin film. Probes were prepared with DIG-11-dUTP mixed in by PCR. Hybridization and coloration followed routine operation procedures of DIG labeled probe. Positive hybridization results were verified with reverse Northern blot.

RESULTSThe positive results from the PCR-Select differential screening Kit improved with DIG labeled probe achieved 90% correspondence verified by reverse Northern blot.

CONCLUSIONThe PCR-Select differential screening Kit improved with DIG labeled probe maintained high specificity while avoiding isotope pollution. It can replace isotope probe completely.

DNA Probes ; Digoxin ; Nucleic Acid Hybridization ; Polymerase Chain Reaction ; instrumentation ; methods ; Reagent Kits, Diagnostic

AIMTo explore the molecular mechanism of heat stress by isolating differentially expressed genes occurred during the course of heat stress and filtrating functional gene segments concerned with heat stress with Suppression Subtractive Hybridization technique.

METHODSThere were 2 groups in the experiment: heat stress group and normal temperature control group. mRNAs were isolated from the liver tissue individually and then transcripted into cDNAs. After cut with endonuclease and ligation with adaptors, SSH was carried out with cDNAs of heat stress group as tester while cDNAs of control group as driver. Products were cloned into T/A vector to form recombined plasmids to transfect competence cells for filtration and appraisal.

RESULTSmRNAs were abundant and of high quality, cDNAs were reverse-transcripted and then cut into segments about 500 bp successfully. Ligation efficiency was satisfied. The subtractive hybridization library was constructed successfully after effective hybridization and 180 segments were primarily isolated from it.

CONCLUSIONConstruction of heat stress differentially expressed genes subtractive library has established the foundation of filtrating heat stress concerned genes, it is of active significance for exploring functional genes that controlled heat stress.

Animals ; DNA, Complementary ; Gene Expression ; Heat Stress Disorders ; genetics ; Liver ; metabolism ; Male ; Nucleic Acid Hybridization ; methods ; Rats ; Rats, Sprague-Dawley ; Transcriptome

OBJECTIVETo observe the effect of 25-hydroxyvitamin D3 on the permeability and ZO-1 expression in normal human airway epithelial cells.

METHODSMTT assay was used to assess the viability of human airway epithelial cell line 16HBE following a 24-hour exposure to different concentrations of 25-hydroxy vitamin D3, and the transepithelial electrical resistance (TER) of the cell monolayer was measured using a Millicell-ERS voltohmmeter. Real-time quantitative RT-PCR was employed to determine the changes of ZO-1 mRNA expression in the cells following the exposures.

RESULTSExposure to 25-hydroxyvitamin D3 resulted in significantly increased permeability of 16HBE cells, but the exspression of ZO-1 showed no obvious changes. 25-hydroxyvitamin D3 at 4×10(-9) mol/L showed the strongest effect in increasing the permeability of cell monolayer.

CONCLUSION25-hydroxyvitamin D3 increases the permeability of normal bronchial airway epithelial cell monolayer in vitro, but this effect is not mediated by upregulation of ZO-1 expression.

Bronchi ; cytology ; metabolism ; Calcifediol ; pharmacokinetics ; pharmacology ; Cell Line ; Cell Membrane Permeability ; drug effects ; Epithelial Cells ; cytology ; metabolism ; Humans ; RNA, Messenger ; genetics ; metabolism ; Zonula Occludens-1 Protein ; genetics ; metabolism

Objective To solve the danger and difficulty in transferring seriously injured victims. Methods The operating principle, construction design, electronic control system and software program flowchart of a robot transfer arm for victim-transfer were introduced.Results and Conclusion The victim didn not have to change their body posture during transfer. The procedure is very simple.A push at only one key is enough,without secondary injury.

OBJECTIVETo investigate the clinical indications of asthma control test (ACT).

METHODSA total of 120 asthmatic patients with a diagnosis in line with the American Thoracic Society criteria and treated for over a month were enrolled in this study. The patients were asked to complete a survey to assess their symptoms and asthma attacks, and ACT evaluation was conducted by physicians familiar with ACT evaluation. The patients were classified into two groups based on the pulmonary function test (positive for bronchodilator test and provocation test) or based on disease severity (mild and moderate-to-severe asthma groups). The effect of ACT evaluation was graded as good (no less than 4 item available for evaluation), fair (2-3 items available) and poor (no more than 1 item). To further analyze the ACT sensitivity in relation to different disease severity, 29 asthmatic patients with an initial diagnosis and BDT positivity were included, and the ACT score of the patients with mild, moderate and severe asthma based on FEV1% were compared.

RESULTSIn patients positive for bronchodilator test, good, fair and poor evaluation effects were found in 48, 15, and 5 cases, as compared to 10, 15, and 27 in those positive for provocation test, respectively, showing significant differences between the two groups (P < 0.001). In mild asthma group, good, fair and poor evaluation effects were found in 12, 15, and 18 cases, respectively, significantly different from those in moderate-to- severe asthma group (50, 21, and 4 cases, P < 0.001). ACT scores showed a positive correlation to FEV1% in 29 patients with positive BDT (r = 0.55, P = 0.003). ACT scores had no significant difference between mild and moderate asthma groups (P > 0.05), but showed significant differences between mild and severe groups (P = 0.009) and between moderate and severe groups (P = 0.008).

CONCLUSIONACT is more suitable for evaluating patients positive for bronchodilator test or with moderate to severe asthma.

Adolescent ; Adult ; Aged ; Asthma ; diagnosis ; physiopathology ; Female ; Humans ; Male ; Mass Screening ; Middle Aged ; Predictive Value of Tests ; Respiratory Function Tests ; Sensitivity and Specificity ; Severity of Illness Index ; Surveys and Questionnaires ; Young Adult

OBJECTIVETo evaluate the time-effect of silica on the expression of lung tissue nitric oxide synthase (NOS) in early inflammatory damage stage of silicotic rat.

METHODSAnimal models were established by direct tracheal instillation of silica into rat lungs. Total NOS and induced NOS (iNOS) activities in bronchoalveolar lavage fluid (BALF) were assayed. The expression of iNOS protein in paraffin-embedded lung sections with Streptavidin/peroxidase (SP) immunohistochemistry were measured by tissue microarray and Image-Pro Plus.

RESULTSMost of the expression of iNOS was in the cytoplasm of macrophages and neutrophils. iNOS integrated optical density (IOD) of lung tissue increased 1.47 x 10(5) and 2.73 x 10(5) more respectively in silicatreated rats 3, 7 days after exposure than in control rats (P < 0.05), and decreased 1.11 x 10(5) more 28 days after exposure (P < 0.01). The activities of iNOS in BALF increased by 0.86, 1.89 and 0.92 U/ml respectively 3, 7, 14 days after exposure (P < 0.05 or P < 0.01). The activities of total NOS in BALF increased by 1.43, 2.05, 2.61 and 2.19 U/ml respectively 1, 3, 7, 14 days after exposure (P < 0.05 or P < 0.01).

CONCLUSIONAfter silica instillation, the iNOS-positive cells in rat lung tissue were mostly macrophages and neutrophils. There is a parabolic changing trend in the level of expression of lung iNOS during 1 - 28 day exposure to silica.

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Female ; Immunohistochemistry ; Lung ; drug effects ; enzymology ; Male ; Models, Animal ; Nitric Oxide Synthase ; analysis ; metabolism ; Rats ; Rats, Wistar ; Silicon Dioxide ; toxicity

OBJECTIVEIt has been known that the intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis in livers. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on the signal passageway of active protein-1 (AP-1).

METHODSIn vitro, HSCs-T6 cell line was treated with Aldo for 10 min, 30 min, 60 min, 120 min and 180 min, and protein expression of Phospho-P42/44 was detected by Western blot. In addition, HSCs-T6 cell line was preincubated for 60 min or not with U0126 (an inhibitor of the MAPK/ERK kinase), and also with antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expression of Phospho-P42/44 was measured by Western blot. DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, expression of alpha1(1) procollagen mRNA was detected.

RESULTSAldo stimulated HSC via extracellular signal-regulated kinase (ERK1/2) pathway. Time course experiments showed that Aldo induced Phospho-P42/44 expression, which was abrogated by U0126, reaching a maximum at 10 minutes, and then declined progressively. NAC inhibited the Phospho-P42/44 expression. EMSA showed that stimulation of HSC by Aldo markedly increased AP-1 DNA binding activity. U0126 markedly reduced AP-1 DNA binding activity induced by Aldo; NAC partly decreased AP-1 activity induced by Aldo. Aldo up-regulated expression of alpha1(1) procollagen mRNA, which was attenuated by U0126 and NAC.

CONCLUSIONStimulation of HSC by Aldo results in activation of AP-1 via ERK1/2 pathway, leading to up-regulation of AP-1 target gene alpha1(1) procollagen mRNA expression.

Aldosterone ; pharmacology ; Cell Line ; Collagen Type I ; biosynthesis ; genetics ; Hepatocytes ; cytology ; metabolism ; Humans ; Mitogen-Activated Protein Kinase 3 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factor AP-1 ; metabolism

A modified protocol for quick and economic treatment of cultured human umbilical vein endothelial cells has been established, which result in high viability of the cells to allow better performance in patch-clamp studies. The electrophysiological properties of Ca (2+)-activated K(+) (KCa) channel of the cultured cells were investigated with a cell-attached configuration. With this modified treatment method, the cultured cells appear fusiform in shape under microscope, and KCa channel currents could be detected readily, suggesting their eligibility for patch-clamp studies.
Cells, Cultured ; Endothelial Cells ; cytology ; physiology ; Humans ; Membrane Potentials ; physiology ; Patch-Clamp Techniques ; methods ; Potassium Channels, Calcium-Activated ; physiology ; Reproducibility of Results