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.

Objective To explore the effects of different flushing and sealing procedures on blood pressure fluc-tuation in patients receiving norepinephrine(NE) via micro-pump. Methods A total of 40 cases of critically ill pa-tients receiving intravenous infusion of NE via micro-pump,were randomly divided into two groups(20 cases in each group) from March to September,2016. For the experimental group,the liquid medicine in central venous catheter was sucked out,followed by flushing or sealing the tube according to conventional operation method. For the control group,conventional operation method was used to flush or seal the tube. The effects of two methods on arterial blood pressure were compared. Results Overall 423 flushing and sealing events were recorded among 40 cases in this study (209 in the experimental group and 214 in the control group). The fluctuation of blood pressure was small in the experimental group,while patients in the control group had significant fluctuation of blood pressure(P<0.05). Conclusion The new method that sucking liquid medicine out of the central venous catheter before flush-ing or sealing the tube followed by flushing or sealing using conventional operation method can reduce the risk of sudden increase in blood pressure for patients receiving small dose infusion with micro-pump.

Guidelines as Topic ; Health Personnel ; Humans ; Occupational Diseases ; prevention & control ; Occupational Health ; legislation & jurisprudence

Objective To investigate the infect capability of recombinant adenovirus mediated PDGF to human umbilical cord derived mesenchymal stem cells (MSCs). Methods The PDGF cDNA sequence was amplified with RT-PCR and constructed recombinant adenovirus vector AdPDGF. The human umbilical cord derived MSCs were isolated and cultured. In vitro AdPDGF infected MSCs with various MOI,and determine the efficiency using FACS and fluorescence microscope. Trypan blue and MTT assay was used to detect cell viability and proliferation. ELISA was used to quantify PDGF secretion. Results Recombinant adenovirus AdPDGF was successfully and constructed,in which infection efficiency to MSCs was dose dependent. The highest efficiency was around 87.36 % when MOI =50,at which cell viability and proliferation wasn' t impaired. Cell viability of uninfected,AdPDGF infected and control virus infected MSCs was (97.8 ±2.3) %,(91.9±4.0) % and (92.8±4.0) %,separately. The cell proliferation was (100±16.8) %,(95.9±12.0) % and (87.5±9.7) %,separately. There was no statistic significance among AdPDGF infected,control virus infected and uninfected MSCs. 48 hrs post infection,PDGF secretion can be measured from infectious MSCs' supernatant. The secretion level was (1.53±0.37),(3.03±0.68) and (5.25±0.92) ng/ml,separately,when MOI= 10,30,50. No PDGF can be detected from MSC-GFP and control MSC group. Conclusion Recombinant adenovirus AdPDGF was constructed and can infect human umbilical cord derived MSC at high efficiency.

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.

Objective To evaluate the clinical value of CT perfusion (CTP) imaging for providing quantitative information about angiogenesis in patients with lung carcinoma and investigate the correlation of CTP enhancement parameters and histological microvessel density (MVD) with lymphatic involvement in peripheral lung carcinoma. MethodsFifty-three patients with pathology-proved peripheral lung carcinoma underwent CT perfusion scan before operation. The enhancement parameters of CTP were calculated based on the time-density curves (TDC) of fist pass phase. All cases were classified into two groups according to pathologic results: tumor with and without lymph node involvement. Two-sample t test was used for the statistics. The ROC curve was used to assess the efficiency of the enhancement parameters of CT perfusion and MVD for predicting lymphatic involvement.Results Tumors with lymph node involvement had significantly higher value of MVD than those without lymph node involvement (64.69±16.34 and 42.67± 16.78, respectively,t=4.84,P<0.01). Tumors with lymph node involvement had significantly higher value of CTP enhancement parameters (PH, M/A, PV) than those without lymph node involvement [PH= (41.79±15.50) and (29.99±10.91) HU,M/A =0.24±0.09 and 0.15±0.06, PV=(2.14±1.09) and (1.27±0.53) ml·min~(-1)·ml~(-1), t=3.21,3.95, 3.66, P<0.01, respectively]. The CTP enhancement parameters (PH, M/A, PV) of lung cancer correlated positively with the MVD, the highest correlation coefficient was between the PV and MVD (r=0.716, P<0.01). MVD and PV had higher values for predicting lymph node involvement in ROC curve analysis.The sensitivity, specificity and accuracy for predicting lymph node involvement were 80.8%, 81.5% and 81.1% or 84.6% ,85.2% and 84.9% respectively if MVD>52/0.74 mm~2 or PV>1.52 ml·min~(-1)·ml~(-1). ConclusionThe CT perfusion PV and histological MVD have good correlation with lymph node involvement in peripheral lung carcinoma and are important predicting parameters before operation.

Objective To investigate the expression of SP 100 protein in ATRA-treated NB4 cells and its effect on pro-liferation in NB4 cells.Methods Q-PCR was employed to measure the expression of SP 100 mRNA;Western blot was used to detect the expression of SP 100 protein; Immunofluorescence was adopted to determine the location of SP100;Cell viability was analyzed by CCK 8;Flow cytometry was used for cell cycle analysis .Results ATRA may induce the expression of mRNA and protein of SP 100.ATRA changes the location of SP100 from a micro-punctate pattern into a punctate nuclear pattern in NB 4 cells.SP100-shRNA promotes the proliferation of NB 4 cells and in-creased the cells in G2/M phase.Conclusions The expression of SP100 was significantly increased in ATRA-treated NB4 cells, and SP100 may be involved in the regulation of proliferation activity of NB 4 cells.

Objective:To develop a learning scale of scientific methods for medical students and to evaluate the reliability and validity of the scale.Methods:Based on the principles of planned behavior theory, the original scale was developed through literature analysis, pre-interview and experts’ consultation. A preliminary survey was conducted among 105 medical undergraduates with random cluster sampling, and the primary scale was formed by exploratory factor analysis. Then, 851 undergraduates were selected as the objects of formal survey, and the formal scale was finally determined by the reliability and validity evaluation and confirmatory factor analysis.Results:The formal scale was composed of 5 latent variables, 9 observed variables and 51 items. The overall Cronbach’s α coefficient and the overall retest reliability were 0.970 and 0.887, respectively. Composite reliability and KMO values of each latent variable were more than 0.8, and the values of average variance extraction were over 0.5. The fitness test ( P=0.155) showed acceptable fitting quality with the main fit indices of χ2/ df=1.405, RMR=0.039, RMSEA=0.022, etc. Conclusion:The learning scale of scientific methods shows satisfactory reliability, validity and fitness, which can be applied as a measuring tool to evaluate the learning behavior of scientific methods for medical students.

Background and purpose: Regional lymph node metastasis was significantly associated with the prognosis of patients with non-small cell lung cancer (NSCLC). This study was designed to compare paclitaxel liposome plus cisplatin (LP) with gemcitabine and cisplatin (GP) in patients with regional lymph node metastasis of advanced NSCLC as a ifrst-line treatment. Methods:A total of 55 patients were randomly assigned to receive either liposomal paclitaxel (175 mg/m2) and cisplatin (75 mg/m2) or gemcitabine (1 000 mg/m2) and cisplatin (75 mg/m2) every 3 weeks. Results:Objective response rate (ORR) of lung primary foci was 37.9%in the LP arm and 30.8%in the GP arm (P>0.05) and the disease control rate (DCR) was 91.3%and 80.8%respectively (P>0.05);ORR of regional metastasis lymph node was higher in the LP arm (44.8%vs 15.4%, P<0.05).There was no signiifcant difference in DCR (93.1%vs 73.1%, P=0.101), although slight trends favoring paclitaxel liposome were seen;There was signiifcant difference in median overall survival (17.0 vs 12.0 months, P<0.05). LP was associated with significantly less thrombocytopenia and gastrointestinal side effects (P<0.05), but no signiifcant difference was observed in hyphemia, leucopenia, hepatotoxicity, renal toxicity and allergic reactions (P>0.05). Conclusion: Liposomal paclitaxel plus cisplatin is superior to gemcitabine plus cisplatin with less toxicity and better tolerated, it deserves further research and clinic application for patients with regional lymph node metastasis of advanced NSCLC.

Objective:To develop a learning scale of scientific methods for medical students and to evaluate the reliability and validity of the scale.Methods:Based on the principles of planned behavior theory, the original scale was developed through literature analysis, pre-interview and experts' consultation. A preliminary survey was conducted among 105 medical undergraduates with random cluster sampling, and the primary scale was formed by exploratory factor analysis. Then, 851 undergraduates were selected as the objects of formal survey, and the formal scale was finally determined by the reliability and validity evaluation and confirmatory factor analysis. SPSS 22.0 and AMOS 22.0 softwares were used for statistical analysis of the measurement data.Results:The formal scale was composed of 5 latent variables, 9 observed variables and 51 items. The overall Cronbach’s α coefficient and the overall retest reliability were 0.97 and 0.89, respectively. Composite reliability and KMO values of each latent variable were more than 0.80, and the values of average variance extraction were over 0.50. The fitness test ( P=0.155) showed acceptable fitting quality with the main fit indices of χ2/ df=1.41, RMR=0.04, RMSEA=0.02, etc. Conclusion:The learning scale of scientific methods shows satisfactory reliability, validity and fitness, which can be applied as a measuring tool to evaluate the learning behavior of scientific methods for medical students.