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Objective To investigate the protective effect of hepatocyte growth factor (HGF) on cultured Sprague-Dawley rat cortical neurons injured through hypoxia/reoxygenation.Methods Primary cultured cerebral cortical neurons were isolated from newborn rots.Neurons were pre-incubated with different concentrations (15,30 and 60 μg/L) of HGF.The cell viability was detected by MTT.Apoptosis was measured by Hoechst 33258 staining and flow cytometer.Lactate dehydrogenase (LDH) and caspase-3 activity were determined by colorimetry.Results Compared with normal group,hypoxia/reoxygenation treatment significantly decreased cell viability,increased LDH activity and the percentage of apoptotic cells.Pretreatment of HGF for 12 h could remarkably reverse the decrease of cell viability and the increase of apoptosis rate in neurons induced by hypoxia/reoxygenation treatment.HGF pre-treatment also attenuated the activity of LDH and caspase-3 in a dose-dependent manner.The effects of HGF could be inhibited by a special PI3K/Akt pathway inhibitor,LY294002.Condusion HGF could attenuate rat cortical neuron injury induced by hypoxia/reoxygenation.The neuroprotective effect of HGF may be related to activating PI3K/Akt pathway,and further suppressing the expression of caspase-3.
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Objective: To identify the different protein expression profiles between human oral squamous cell carcinoma (OSCC) and normal oral mucosa tissues, and provide experimental data for further study of the development mechanism of OSCC. Methods: 10 cases of OSCC and paired normal oral mucosa tissues were collected and analyzed through two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Results: (1) The average protein spots of OSCC were 2 325±390, while that of normal oral mucosa tissues were 2 487±281. (2) 29 differential protein spots were found between OSCC and normal oral mucosa. Moreover, these protein spots were all down- regulated in OSCC compared with normal oral mucosa. Among these spots, 3 were identified as fibrin beta, triosephosphate isomerase (TIM) and unknown protein through mass spectrometry and bioinformation. Conclusion: Down-regulation of fibrin beta, Triosephosphate isomerase(TIM) and unknown protein are found in the development of OSCC and the mechanism needs further study.
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BACKGROUND:FGF2,TGFβ/activin/nodal and IGF signal pathways are necessary for keeping functions of human embryonic stem calls,but there was no report concerning whether addition of exogenous basic fibroblast growth factor,transforming growth factor β and insulin can maintain self-renewal of human embryonic stern cells.OBJECTIVE:To establish a feeder layer-and serum-free culture system of human embryonic stem cells.DESIGN,TIME AND SETTING:The cytological in vitro study was performed at the Kunming Animal Institute of Chinese Academy of Sciences from September 2007 to February 2009.MATERIALS:Pregnancy 12.5 or 13.5-day ICR strain mica (clean grade) were provided by the Animal Center of Kunming Medical College.Human embryonic stem cell line BG02 was purchased from Bresagen,USA.METHODS:BG02 cells were digested,centrifuged,resuspended in feeder layer-free medium,and then incubated for 5-7 days.Differentiated human embryonic stem cells were removed.Dispase was added for digestion.Samples were cut into blocks,centrifuged,resuspended,and then incubated at 1:3 in a 4-well plate coated with laminin in feeder layer-free and serum-free medium,supplemented with 80% DMEM/F12,20% KSR,2 mmol/L glutamine,1% non-essential amino acid,0.1 mmol/L β-mercaptoethanol,insulin-transferrin-selenium (ITS) (×1),10~6 U/L penicillin,100 mg/L streptomycin,4 μg/L basic fibroblast growth factor (bFGF) and 0.12 μg/L transforming growth factor-β1 (TGF-β1).ICR fetus mica were sterilely obtained to culture mouse embryonic flbroblasts by tissue pancreatin digestion method.These cells were incubated in a 6-well plate coated with 0.1% gelatin.Human embryonic stem cell line BG02 cultured on mouse embryonic fibroblasts feeder layers was transferred to laminin-coated plates in serum-free medium containing bFGF,TGFβ1 and ITS.MAIN OUTCOME MEASURES:The morphology of BG02 cells in feeder layer-and serum-free condition was observed.The specific molecular markers of human embryonic stem cells were determined by immunohistochemical staining.The karyotype and differentiation ability in vitro of BG02 cells were analyzed.The differences in the proliferation and the differentiated rate of BG02 clumps in feeder layer-and serum-free condition or mouse embryonic fibroblast feeder layer condition were compared.RESULTS:BG02 calls had been continuously cultured for at least 20 passages in feeder layer-and serum-free culture condition.BG02 clones in this culture system had typical human embryonic stem cell morphology.BG02 cells all expressed SSEA-4,SSEA-3,TRA-1-60,TRA-1-81,Oct-4,but did not express SSEA-1.After 20 days of culture,BG02 cells had the capacity to differentiate into derivatives of embryonic endoderm,mesoderm,and ectoderm.Differentiated cells could express alpha-fetoprotein,nestin,α-actin.At passage 20,call karyotype was normal (46XY).The growth of BG02 cells cultured in feeder layer-and serum-free condition was more slowly compared with mouse embryonic fibroblast feeder,and doubling time was significantly prolonged (P ＜ 0.05).Differentiation rate of the colonies was significantly increased (P ＜ 0.05).CONCLUSION:A feeder layer-and serum-free condition for culture of BG02 cells has been established.The addition of bFGF,TGFβ1 and ITS can maintain the self-renewal of BG02 cells.
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BACKGROUND: Whether human embryonic stem cells (hESCs) cultured on different feeder layers can maintain identical or similar characteristics remains unclear.OBJECTIVE: To compare the characteristics of hESCs cultured on human- and mouse- origin feeder layers.DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Kunming Institute of Zoology, Chinese Academy of Sciences between September 2007 and February 2009.MATERIALS: Two ICR pregnant mice with 12.5-13.5 embryonic days were provided by Animal Center of Kunming Medical College. Immortalized human adult fibroblast (HAFi) cell line was presented by School of Medicine, Johns Hopkins University (USA). hESCs line was provided by Kunming Institute of Zoology, Chinese Academy of Sciences.METHODS: Mouse embryonic fibroblasts (MEF) were harvested from ICR mice by trypsinization method. HAFi were conventionally cultured. After γ ray treatment, two kinds of cells were incubated on 6-well gelatin-coated plate with density of 2.5×10~4/cm~3. hESCs were cultured on HAFi or MEF feeder cells containing β-mercaptoethanol DMEM/F12 and basic fibroblast growth factor.MAIN OUTCOME MEASURES: Morphology, expressions of specific molecular markers, Oct-4 positive rate, as well as cell doubling time of hESCs cultured by two methods were compared.RESULTS: ①BG02 cells on MEF and HAFi shared the similar morphology and characteristic pluripotency markers, which expressed SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 and Oct-4, but not SSEA-1. However, the proportion of positive Oct-4 cells in hESCs colonies maintained on MEF was lower than that of HAFi (P < 0.05) with shorter doubling time (P < 0.05).CONCLUSION: hESCs cultured on MEF and HAFi represent some differences in the growth and pluripotency characteristics.
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Teaching is an art,which requires teachers to have high profession level and better ability of language expression.To stress teaching techniques is the request of higher medical science education.It is worth summarizing and thinking seriously.This article summarized several aspects of teaching techniques,essence.In addition,it explained the significance of teaching techniques.
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Objective:To find biomarkers for oral lichen planus by comparing differential expressing proteins. Methods:10 cases of oral lichen planus and normal oral mucosa tissues were collected.Total protein was extracted; differential proteome profiles were established and analyzed by two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Results:(1)The well-resolved,reproducible 2-DE patterns of oral lichen planus and normal oral mucosa were obtained. The results showed that average protein spots were 1 576?67 and 1 608?73 in oral lichen planus and normal oral mucosa respectively, (2) The 13 differential protein spots were identified by Imaging Master 2D image analysis software between oral lichen planus and normal oral mucosa. There were 7 protein spots in oral lichen planus were higher than those in normal oral mucosa, 6 protein spots in oral lichen planus were lower than those in normal oral mucosa. 10 differential expressing proteins were analyzed by mass spectrometry and bioinformation. 4 of them were well characterized including manganese superoxide dismutase (Mn-SOD), Annexin I, vimentin and unknown proteins. Conclusion:Differential expression proteins might be candidate biomarkers for diagnosis of oral lichen planus;and proteomic technique is valuable for screening the diagnostic biomarkers.