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, Volume 41 Issue 4 Previous Issue    Next Issue
Embryonic stem cells and regeneration-promoting drug research & development
Journal of ZheJiang University(Medical Science), 2012, 41(4): 353-358.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.001
Abstract( 376 )   HTML (   PDF(1071KB)( 392 )
Expression of Junctophilin 1 during cardiogenesis of mouse embryonic stem cells and rat embryos
Journal of ZheJiang University(Medical Science), 2012, 41(4): 359-365.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.002
Abstract( 495 )   HTML (   PDF(3711KB)( 297 )
Objective: To investigate the expression of Junctophilin 1 (JP1) in cardiogenesis of mammalian. Methods: Cardiac differentiation of embryonic stem cells (ESCs) was generated by hanging drop method. Fetal heart was obtained from the rats aged d 14-20 of gestation. The expression of JP1and JP2 during cardiogenesis of ESCs and rat embryos was analyzed by RT-PCR or Western blotting. Immunofluorescence staining was employed to reveal the distribution of JP1 and JP2 in embryoid body (EB), probing for merging of JP1and JP2 and cardiac sarcomeric α-Actinin or Troponin-T. Percentage of JP1and JP2-positive staining cells was analyzed quantitatively by FCS on d17.
Results: JP1 mRNA was up-regulated at the early stage (d 5-11) and then decreased. The expression of JP1 protein was up-regulated at the early stage (d 7-9) , then decreased gradually and disappeared after d 15. While JP2 gene and protein expression increased in a time-dependent manner during cardiogenesis of rat embryos. The results of immunofluorescence staining showed that there was a parallel co-localization of JP2 with Troponin-T or α-Actinin on d17, while JP1 failed to express in the sarcomeric positive area at the same time point. Furthermore, FCS analysis showed that about 16.59% of cells were JP2-positive, while no cells were stained positively for JP1 in d17 EBs.
Conclusions: JP1 gene is expressed during the whole process of cardiogenesis, while JP1 protein only appears on the early stage. The expression of JP1 in cardiogenesis of ESCs is consistent with that of rat embryos.
Expression of metabotropic glutamate receptor 4 in cardiomyocytes differentiated from mouse embryonic stem cells in vitro
Journal of ZheJiang University(Medical Science), 2012, 41(4): 366-372.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.003
Abstract( 439 )   HTML (   PDF(2685KB)( 369 )
Objective: To investigate the expression of metabotropic glutamate receptor 4 (mGluR4) in cardiomyocytes differentiated from mouse embryonic stem cells (ES cells).
Methods: ES cells were differentiated into cardiomyocytes with hanging-drop cultures. Retinoic acid (RA) and dimethyl sulfoxide (DMSO) were used as positive and negative controls, respectively. The co-expression of cardiac sarcomeric protein (α-actinin or troponin-T) and mGluR4 were verified by immunocytochemistry and flow cytometry analysis. The mRNA and protein expressions of mGluR4 were verified by RT-PCR and Western blot analysis, respectively. Meanwhile, the expression of mGluR4 in prenatal mouse heart was also examined.
Results: mGluR4 was expressed in both mouse ES cells and ES cell-derived cardiomyocytes. The level of mGluR4 protein expression decreased during the maturation of the cardiomyocytes. The co-expression rate of mGluR4 and Troponin T in the beating embryoid bodies (EBs) was only(3.00±1.00)%. On the other hand, mGluR4 gene and protein expressions showed remarkable down-regulation in the development of mouse fetal heart, which was not detected in mouse adult heart.
Conclusion: The expression of mGluR4 is down-regulated in the cardiomyocyte differentiation of ES cells. The trend of expression is consistent with that in the prenatal mouse heart development.
Phenotype-based primary screening for drugs promoting neuronal subtype differentiation in embryonic stem cells with light microscope
Journal of ZheJiang University(Medical Science), 2012, 41(4): 373-380.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.004
Abstract( 479 )   HTML (   PDF(6108KB)( 335 )
Objective: To set up a platform for phenotype-based primary screening of drug candidates promoting neuronal subtype differentiation in embryonic stem cells (ES) with light microscope.
Methods: Hanging drop culture 4-/4+ method was employed to harvest the cells around embryoid body (EB) at differentiation endpoint. Morphological evaluation for neuron-like cells was performed with light microscope. Axons for more than three times of the length of the cell body were considered as neuron-like cells. The compound(s) that promote neuron-like cells was further evaluated. Icariin (ICA, 10-6mol/L) and Isobavachin (IBA, 10-7mol/L) were selected to screen the differentiation-promoting activity on ES cells. Immunofluorescence staining with specific antibodies (ChAT, GABA) was used to evaluate the neuron subtypes.
Results: The cells treated with IBA showed neuron-like phenotype, but the cells treated with ICA did not exhibit the morphological changes. ES cells treated with IBA was further confirmed to be cholinergic and GABAergic neurons.
Conclusions: Phenotypic screening with light microscope for molecules promoting neuronal differentiation is an effective method with advantages of less labor and material consuming and time saving, and false-positive results derived from immunofluorescence can be avoided. The method confirms that IBA is able to facilitate ES cells differentiating into neuronal cells, including cholinergic neurons and GABAergic neurons.
Establishment of optimized neuronal differentiation-promoting model derived from P19 embryonic carcinoma cells
Journal of ZheJiang University(Medical Science), 2012, 41(4): 381-385.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.005
Abstract( 445 )   HTML (   PDF(2488KB)( 309 )
Objective: To establish an optimized primary drug screen model of neuronal differentiation using P19 embryonal carcinoma cells.
Methods: The final concentration of retinoid acid (RA), days of suspension culture, manner of adherent culture, suitable cell density and adherent culture medium were tested, respectively. Two stages of neuronal differentiation were examined based on morphological changes and immunocytochemistry analysis of neuronal specific protein β-tubulin Ⅲ.
Results: On d 8 of differentiation culture, neuron-like cells were observed with final concentration of 1 μmol/L RA. Neuron-like network was formed on d 16 of neuronal differentiation. β-tubulin Ⅲ was positively stained on both stages, indicating P19 cells were differentiated into neurons.
Conclusion: The model using RA to induce P19 embryonic carcinoma cells to differentiate into neuron-like cells has been successfully established, which may provide a rapid, phenotypic cell-based platform for primary screening of neurogenesis-promoting drugs.
Construction of directional differentiation model from mouse embryonic stem cells to leydig-like cells in vitro
Journal of ZheJiang University(Medical Science), 2012, 41(4): 386-392.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.006
Abstract( 476 )   HTML (   PDF(2595KB)( 339 )
Objective: To construct a directional differentiation model from mouse embryonic stem cells into leydig-like cells in vitro.
Methods: Mouse ES-D3 cells were transfected with plasmid containing steroidogenic factor 1 (SF-1) gene, then treated with RA and 8Br-cAMP, while the cells transfected with empty plasmid were used as the negative controls. The morphology of leydig-like cells differentiated from ES-D3 cells was observed with light microscopy. The expression levels of StAR, P450scc and 3β-HSD were detected by RT-PCR, Western Blot and fluorescence microscopy analysis in leydig-like cells derived from the ES cells.
Results: ES-D3 cells were transfected with plasmid containing SF-1 gene successfully, and SF-1 was expressed 24 h after transfection. The SF-1-transfected ES-D3 cells were induced by RA and 8Br-cAMP to differentiate into leydig-like cells. The differentiated cells showed spindle shape with tentacles, which expressed the specific protein marker for leydig cells 3β-HSD1 and P450scc. Meanwhile, in these leydig-like cells, the expression of StAR increased compared with control group. 3β-HSD1, P450scc and StAR were not detected in negative control group.
Conclusion: When the ES-D3 cells are transfected with SF-1 plasmid and then treated with RA and 8Br-cAMP, the cells are able to differentiate into leydig-like cells, indicating that the model of directional differentiation of ES cells into leydig-like cells has been constructed successfully.
Ginsenoside Rg1 antagonizes β-amyloid peptide-induced apoptosis in primarily cultured rat neurons via mitochondrial pathway
Journal of ZheJiang University(Medical Science), 2012, 41(4): 393-401.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.007
Abstract( 383 )   HTML (   PDF(7233KB)( 304 )
Objective: To assess the neuroprotective effects of ginsenoside Rg1 against β-amyloid peptide (Aβ25-35) -induced apoptosis in primarily cultured rat cortical neurons.
Methods: Primarily cultured cortical neurons were obtained from embryonic (E18d) rat fetus and maintained in neurobasal medium for 7d. Primary neurons pretreated with 1 μmol/L, 10 μmol/L or 20 μmol/L Rg1 for 24 h were challenged with 10 μmol/L Aβ25-35 for 72 h. Morphological changes of neurons were evaluated; mitochondrial membrane potential (ΔΨm) was measured; with JC-1 staining and the expression of neural apoptosis-related proteins was detected by Western blot analysis.
Results: Exposure to Aβ25-35 for 72 h caused serious neural cell insults. A pretreatment with Rg1 significantly reduced Aβ25-35-induced cell death in a dose-dependent manner, with a maximal effect (-90%) obtained at 20 μmol/L. The JC-1 staining results demonstrated the loss of ΔΨm after Aβ25-35 treatment, while Rg1 maintained the normal level of ΔΨm. A series of mitochondrion-mediated apoptotic events happened after Aβ25-35 treatment, such as decrease of Bcl-2/Bax, release of cytochrome C and activation of caspase 9 and caspase 3, which were all blocked by Rg1 pretreatment. Both estrogen receptor (ER) antagonist ICI182,780 and glucocorticoid receptor (GR) antagonist RU486 blocked the antiapoptotic effects of Rg1.
Conclusions: Ginsenoside Rg1 protects primary cultured rat cortical neurons from Aβ25-35-induced injury, which may be associated with mitochondrion-mediated antiapoptosis pathway.
Effect of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus
Journal of ZheJiang University(Medical Science), 2012, 41(4): 402-409.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.008
Abstract( 481 )   HTML (   PDF(2398KB)( 318 )
Objective: To investigate the effects of chronic lead exposure on expression of autophagy-associated proteins in rat hippocampus.
Methods: SD rats were randomly divided into three groups: control group was given distilled water, lead-exposed groups were given 0.5 g/L (low-dose) or 2.0 g/L(high-dose) lead acetate solution in drinking water. The rat pups started to drink the lead content water until 60 d maturity. The lead contents in blood and brain samples were analyzed by graphite furnace atomic absorption spectrophotometry. The expressions of Beclin 1, LC3, LAMP2 and cathepsin B proteins were detected by Western blot and immunohistochemistry.
Results: Compared with control group, the contents of lead were significantly higher in blood and hippocampus samples in chronic lead-exposed rats (P<0.01). Western blot showed that the expression of Beclin 1 and LC3-II/LC3-I increased significantly in high dose lead-exposed group compared with control group (P<0.05 or P<0.001). The confocal laser immunostaining results demonstrated that increased immunofluorescence staining of cathepsin B in hippocampal neurons compared with control animals.
Conclusion: The disturbance of autophagy-lysosome signaling molecules might be partially contribute to neurotoxicity of chronic lead exposure.
Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species
Journal of ZheJiang University(Medical Science), 2012, 41(4): 410-417.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.009
Abstract( 413 )   HTML (   PDF(3092KB)( 313 )
Objective: To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species.
Methods: The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagenoblast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P2X1, P2X2, P2X3, P2X4, P2X5, P2X6 and P2X7 were determined with Western blot assay.
Results: β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P2X1 receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P2X5 receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets.
Conclusion: There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospire-induced injury in different host cells.
Development of datasets for basic medical information system
Journal of ZheJiang University(Medical Science), 2012, 41(4): 418-424.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.010
Abstract( 676 )   HTML (   PDF(984KB)( 305 )
Objective: To develop dataset for basic medical information system.
Methods: All items of basic medical information system dataset were drafted by literature review, brain storm and group discussion. A Delphi study was conducted to evaluate and adjust the candidate items; then items were finalized based on comprehensive scores.
Results: The final dataset developed through two-round Delphi study contained 20 first-level items, 102 second-level items, 40 third-level items and 88 fourth-level items. The response rates were 71.79% and 92.86% in round 1 and 2, respectively. The authority coefficient was both 0.77 and the coordination coefficient of the specialists′ opinion was 0.239 and 0.516, respectively.
Conclusion: The dataset for basic medical information system determined from Delphi study is credible and can be used for development of related software.
Effect of Colquhoumai root tablet on IL-2 and IFN-γ mRNA expression in rats with experimental allergic encephalomyelitis
Journal of ZheJiang University(Medical Science), 2012, 41(4): 425-429.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.011
Abstract( 405 )   HTML (   PDF(2070KB)( 330 )
Objective: To investigate the effect of Colquhoumai root tablet on IL-2 and IFN-γ mRNA expression in experimental allergic encephalomyelitis (EAE) of rats.
Methods: The allergic encephalomyelitis model was established in Wistar rats by immunization with myelin basic protein of spinal cord of guinea pig and complete Freund′s adjuvant.The rats in treatment group received Colquhoumai root tablet (300 mg·kg-1,BID).The symptom of EAE was observed; pathological feature and myelin of brain and spinal cord were detected with HE stain and Loyez′s stain, respectively.The expressions of IL-2 and IFN-γ mRNA were assayed by RT-PCR.
Results: No EAE symptoms were developed in treatment group,the expressions of IL-2 and IFN-γ mRNA were 0.345±0.032 and 0.353±0.023,which were significantly lower than those of model group (P<0.01).The histopathologic examinations revealed that less inflammation cells around vessels and demyelization in white matter of brain and spinal cords were observed in treatment group than in model group.
Conclusion: Colquhoumai root tablets are effective in treatment of EAE of rats,which may be associated with inhibition of the expression of IL-2 and IFN-γ mRNA.
A method for simultaneous assay of propulsion and absorption in small intestine
Journal of ZheJiang University(Medical Science), 2012, 41(4): 430-433.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.012
Abstract( 331 )   HTML (   PDF(2014KB)( 332 )
Objective: To develop a method for simultaneous assay of propulsion and absorption in small intestine.
Methods: The mice were administrated through gastric tube with mixed reagents containing 0.12% phenol red, D-xylose (1.25%, 2.5% and 5%) and 15% gelatin. The influence of phenol red on D-xylose absorption and the influence of D-xylose on small intestine propulsion rate were investigated by measuring serum concentration of D-xylose with phloroglucinol method.
Results: At 10 min, no significant difference was found between 5% D-xylose mixed reagent group and 5% D-xylose control. At 15 min, small intestine propulsion rate in 5% D-xylose mixed reagent group, but not in 2.5% and 1.25% D-xylose mixed reagent groups, was significantly higher than in phenol red control (P<0.05).
Conclusion: Gastric administration of mixed reagent containing 0.12% phenol red, 5% D-xylose and 15% gelatin can simultaneously assay propulsion and absorption of small intestine in mice.
Screening and identification of high-yield poly(β-malic acid) bacterial strain
Journal of ZheJiang University(Medical Science), 2012, 41(4): 434-440.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.013
Abstract( 403 )   HTML (   PDF(1772KB)( 301 )
Objective: To isolate and identify the high-yield poly-malic acid (PMLA) bacterial strains from the nature.
Methods: Samples were collected and cultured. The high-yield PMLA bacterial strains were screened through morphological observation, qualitative PMLA tests by HPLC and ITS sequence analysis on the isolated bacterial strains.
Results: A high-yield PMLA strainⅡ04 was isolated, the yield of PMLA of the strain reached to 26.23g/L in the rotary shaker at 25℃ for 7d. From morphological observation and ITS sequences analysis, the strain belonged to Aureobasidium pullulans, and named as Aureobasidium pullulans ZUCC-41. Conclusion: A high-yield bacterial strain has been isolated from the nature and identified to be Aureobasidium pullulans.
Preparation of diazepam transdermal gel and its bioavailability
Journal of ZheJiang University(Medical Science), 2012, 41(4): 441-444.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.014
Abstract( 449 )   HTML (   PDF(1319KB)( 585 )
Objective: To prepare diazepam transdermal gel and to assess its bioavailability.
Methods: Using Carbopol 934 as a gel matrix, the diazepam transdermal gel was prepared with glycerol as the humectant and azone as penetration enhancer. The penetration rate of diazepam through excised rabbit skin was measured by Franz diffusion cell and HPLC method. Using diazepam tablets as control, the relative bioavailability of diazepam gel was determined in rabbits.
Results: The transdermal flux of diazepam gel was 39.26 g/cm2/h and the bioavailability of diazepam gel was 36.25%.
Conclusion: Diazepam gel prepared in the study would be developed as a novel transdermal preparation.
Characteristics of pulmonary valve annular motion identified by quantitative tissue velocity imaging in children with pulmonary hypertension
Journal of ZheJiang University(Medical Science), 2012, 41(4): 445-449.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.015
Abstract( 383 )   HTML (   PDF(2412KB)( 327 )
Objective: To investigate the characteristics of pulmonary valve annular motion by quantitative tissue velocity imaging (QTVI) in children with pulmonary hypertension.
Methods: The pulmonary valve annular motion was assessed by QTVI in 32 children with pulmonary hypertension and 32 healthy children. The QTVI sample volume was set at the point of pulmonary valve annulus to acquire speed-time curve and the parameters from the views of parasternal aortic short-axis or subxiphoid right ventricular outflow long-axis. The parameters of pulmonary valve annular motion of children with pulmonary hypertension were compared to those of normal children.
Results: The speed-time curve of pulmonary valve annulus was similar with that of tricuspid annulus in normal children. Compared to normal children, the ratio of Ea/Aa (the velocity parameter of pulmonary valve annular motion) was significantly lower in children with pulmonary hypertension (0.68±0.36 vs 1.18±0.43,P<0.001); and the value of QTVI-Tei index at the pulmonary annulus was significantly higher (0.82±0.34 vs 0.37±0.05,P<0.001). The QTVI-Tei index was positively correlated with the resistance of pulmonary vessel(r=0.556,P<0.001).
Conclusions: The ratio of Ea/Aa is decreased, the value of QTVI-Tei index is increased and QTVI-Tei index is positively correlated with the resistance of pulmonary vessel in children with pulmonary hypertension.
Echocardiographic diagnosis of partial anomalous pulmonary venous connection
Journal of ZheJiang University(Medical Science), 2012, 41(4): 450-452.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.016
Abstract( 415 )   HTML (   PDF(1399KB)( 1318 )
Objective: To investigate partial anomalous pulmonary venous connection (PAPVC) with echocardiography.
Methods: The right ventricular volume overload was detected by routine echocardiography in 37 child patients, who underwent further echocardiography to find the abnormal locations of pulmonary vein opening at superior, inferior vena cava and right atrium. The ultrasound results were compared with surgical findings.
Results: In 30 patients the ultrasound diagnosis was consistent with surgery results, 7 were misdiagnosed by ultrasound with a detective rate of 81.1%. All 37 PAPVC patients presented varying degrees of right heart enlargement; PAPVC combined with atrial septal defect (ASD) was found in 34 cases.
Conclusions: The possibility of PAPVC should be considered when unexplained right heart volume overload was detected by echocardiography. Superior, inferior vena cava and right atrium should be inspected when the pulmonary veins were not seen in echocardiography.
Nrf2 as a chemoprevention target in gastrointestinal carcinoma
Journal of ZheJiang University(Medical Science), 2012, 41(4): 453-463.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.017
Abstract( 449 )   HTML (   PDF(1799KB)( 404 )
Gastrointestinal tract carcinoma is one of the leading causes of cancer-related death in China. Chemoprevention has been considered as a potential approach to control this type of disease. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a redox-sensitive transcription factor that protects cells from oxidative/electrophilic stresses by activating the expression of a battery of cytoprotective genes through the antioxidant response element (ARE). Recently, Nrf2 has emerged as a novel target for chemoprevention. Several natural or synthetic chemicals, which activate Nrf2/ARE signaling pathway, have showed effectives in animal models, and promises in many ongoing clinical trials. This review summarizes the recent findings on the regulation of Nrf2/ARE signaling pathway, and the developments in both preclinical and clinical studies.
Type I interferon and bacterial infection
Journal of ZheJiang University(Medical Science), 2012, 41(4): 464-468.   https://doi.org/10.3785/j.issn.1008-9292.2012.04.018
Abstract( 761 )   HTML (   PDF(988KB)( 364 )
Interferons (IFNs) are cytokines playing an important role in immune responses. Interferons are classified into two distinct types according to specific interferon receptors (IFNR). Type Ⅰ IFNs include IFN-α and IFN-β, whereas IFN-γ is type Ⅱ IFN. It is well known that type Ⅰ IFNs have important roles in the host defense against viruses through activation of interferon receptor A (IFNAR). However, many recent studies have also demonstrated that type Ⅰ IFNs have effects on immune responses to bacterial infection. This review focuses on the immune regulation of type Ⅰ IFN-mediated signal pathways in bacterial infections such as listeria monocytogenes, streptococcus, mycobacterium tuberculosis, bacillus anthracis, legionella, pseudomonas aeruginosa and others.
18 articles