BACKGROUND:Although the methods of mechanical scrapping,tissue block transplantation,and enzyme digestion to culture endothelial cells have been quite mature,it has been constantly updated.OBJECTIVE:To explore the culture and identification of rabbit aortic endothelial cells.METHODS:Aorta was derived from one-week-old New Zealand rabbit.Following removing the adventitia,the endothelium was digested in the mixture of 2 g/L type Ⅰ collagen,2 9/L type Ⅱ collagen,2 g/L type Ⅳ collagen,and 2 g/L type Ⅴ collagen (1:1:1:1)for 20 minutes,and the digestionwas stopped with culturemedium(1:1).Cells in the endothelium were lightly scraped to make cell suspension,which was centrifuged to precipitate cells with DMEM culture medium containing 20%fetal bovine serum,1 μg/L VEGF,2 μg/L bFGF,and 6 U/L gentamicin.The cells were then blown into single cells and the culture liquid was changed every 48 hours.The cells were passaged according to the ratio of 1:2.Inverted phase contrast microscope was used to observe cell culture;immunohistochemistry and immunofluorescence were used to determine Ⅷ-related antigen;electron microscope was used to detect Weibel-Paladed body.RESULTS AND CONCLUSION:Ⅷ-related antigen and electron microscopy confirmed that the primary and 5-passage endothelial cells were successfully cultured,suggesting that rabbit aortic endothelial cells were cultured into cell line.Ⅷ-related antigen and electron microscopy determined that a combination with W-P body was a good method to determine endothelial cells.

OBJECTIVE To investigate the delayed cytotoxicity effect of chlorpyrifos (CPF) with?drawal on primary hippocampal neurons. METHODS Hippocampal neurons were prepared from SD rat fetuses on the 17th day of gestation. Seven days after culture,neurons were exposed to CPF 10 and 30 μmol · L-1,respectively,for 72 h or for 48 h followed by CPF withdrawal for 24 h. CCK-8 kit and neuronal nuclei(NeuN), 5-bromodeoxyuridine(BrdU) and β Ⅲ tubulin immunofluorescence expression methods were used to evaluate the cell viability. RESULTS Compared with normal control, no significant cytotoxicity was found after CPF 72 h continuous exposure. However,CPF 48 h expo?sure followed by CPF withdrawal for 24 h induced evident cytotoxicity. The amount of BrdU positive and β Ⅲ tubulin positive hippocampal neurons were both decreased significantly(P<0.05),and cell survival and viability reduced after CPF withdrawal. CONCLUSION CPF exposure withdrawal can induce more seriously delayed cytotoxicity than continuous exposure in rat primary hippocampal neurons.

Objective:To explore the DNA methylation patterns and methylation differential genes of patients with coal-burning-borne endemic fluorosis, and to provide a basis for study of the pathogenesis of fluoride-induced body injury.Methods:A case-control study was conducted in Shuicheng County, Liupanshui, Guizhou Province, ten patients with severe fluorosis were selected as the fluorosis group in Douqing Township, where people burning high fluorine coal in open range all year round; and ten people without fluorosis phenotype were selected as the control group in Huaga Township, where firewood was the main fuel. Peripheral blood samples were collected from the two groups of people. Reduced representation bisulfite sequencing (RRBS) technique was used to detect the whole genome DNA methylation pattern ( n = 4) and DNA differentially methylated region (DMR), the DMR differential degree (log 2Ratio) and KEGG pathway enrichment analysis were used to screen the methylation differential genes, and real-time PCR was used to verify the mRNA expression levels of the candidate methylation differential genes( n = 10). Results:The methylation pattern analysis results showed that the methylation levels of all C bases in the genome DNA of the fluorosis group and the control group were (61.53 ± 0.59)% and (62.48 ± 1.53)%, respectively; among them, the methylated levels at CG sites were (63.75 ± 0.65)%, (64.36 ± 1.01)%, at CHG sites were (13.79 ± 0.72)%, (16.69 ± 4.06)%, and at CHH sites were (25.12 ± 1.72)%, (29.77 ± 3.97)%. Compared with the control group, patients in the fluorosis group had 1 000 DMR distributed on different autosomes; and the chromosome 19 was the most with 104 segments. There were 978 DMR-related genes, including 265 hypermethylation genes and 713 hypomethylation genes; KEGG pathway enrichment analysis showed that methylation differential genes were mainly involved in cell metabolism, cancers, and phosphatidylinositol 3-kinase-protein kinase B (PI3K-AKT) and other signaling pathways; combined with the differential degree of DMR, the hypermethylated succinate dehydrogenase complex flavoprotein subunit A pseudogene 3 (SDHAP3, log 2Ratio = 3.487) and hypomethylated nuclear factor κB inhibitor kinase regulatory subunit γ (IKBKG, log 2Ratio =-4.436) were selected as the candidate genes. There were statistically significant differences in the mRNA expression levels of SDHAP3 (0.54 ± 0.08, 1.00 ± 0.00) and IKBKG (1.32 ± 0.39, 1.00 ± 0.00) between fluorosis group and control group ( F = 22.94, 15.09, P < 0.01 or < 0.05). Conclusion:There are a large number of methylation differential genes in the genomes of patients with coal-burning-borne endemic fluorosis and controls, the hypermethylated SDHAP3 and hypomethylated IKBKG may be involved in fluoride induced body injury.

Objective To explore the outpatients′experience of caring and provide references for improving their experience of medical care.Methods The phenomenology research method was adopted in this study,and twenty-one outpatients were subject to an in-depth interview to learn their outpatient experiences.Results Ten themes of experience with caring were extracted as follows:passionate and patient,careful and considerate,respect for others,offers to help,encouraging and comforting,apologies and courteously,humor,truthful considerations,conscientious and responsible,and a caring environment setup.Five themes of experience without caring were extracted as follows:impatient communication, careless seeing of outpatients,delayed service,passive reminders,and facilities without enough details. Conclusions The outpatients may have abundant experiences of caring at the outpatient clinic,also lack of caring sometimes as well.Outpatient managers should keep an eye on outpatients′needs and satisfaction of caring from time to time,to improve the quality of outpatient service.

Thin endometrium is an important factor influencing the in vitro fertilization and embryo transfer, and there is a lack of effective therapy in the treatment of thin endometrium.The aim of this study was to explore a stable animal model of effective thin endometrium, and to promote the research on thin endometrium pathogenesis and provide experimen-tal basis of treatment.To analyze a variety of establishments of endometrial damage animal model reported in the domestic and foreign literatures,it is concluded that perfusing 95% ethanol into uterine cavities of rats can establish a rat model of thin endometrium,and put forward some experience and methods for its improvement.

Objective To compare the long-term efficacy between two radiochemotherapy regimens for locally advanced nasopharyngeal carcinoma (NPC):intensity-modulated radiotherapy with concurrent chemotherapy (CCRT) versus neoadjuvant chemotherapy (NACT) followed by CCRT.Methods A retrospective analysis was performed on the clinical data of 278 patients with locally advanced NPC who were admitted to our hospital from 2001 to 2008.Of the 278 patients,133 received CCRT,and 145 received NACT followed by CCRT (NACT + CCRT).Results The follow-up rate was 96.6％.The 5-year overall survival (OS),distant metastasis-free survival (DMFS),recurrence-free survival (RFS),and progression-free survival (PFS) were 78.1％,78.0％,90.6％,and 72.0％,respectively.There were no significant differences between the CCRT group and NACT + CCRT group in 5-year OS (79.9％ vs.76.4％,P =0.443),DMFS (77.1％ vs.78.9％,P=0.972),RFS (91.6％ vs.89.8％,P=0.475),and PFS (71.6％ vs.72.2％,P=0.731).Subgroup analysis showed that compared with CCRT,NACT + CCRT did not significantly improve 5-year RFS in T3-4N0-1 patients (90.7％ vs.86.9％,P=0.376) and did not significantly improve 5-year DMFS in patients with advanced N-stage disease (57.6％ vs.69.7％,P =0.275).There were significantly higher numbers of individuals with neutropenia,decrease in hemoglobin,and upper gastrointestinal reactions in patients treated with NACT + CCRT than in those treated with CCRT (100 vs.52,P=0.000;64 vs.35,P=0.010;90 vs.63,P=0.044).Conclusions Compared with CCRT,NACT + CCRT does not significantly improve the prognosis in patients with locally advanced NPC and leads to significant increases in grade ≥ 3 toxicities (neutropenia,decrease in hemoglobin,and upper gastrointestinal reactions).The role of NACT in the treatment of locally advanced NPC needs further study

Objective To evaluate the value of two-dimensional speckle tracking echocardiography (2D-STE) in assessing left ventricular systolic function,diastolic function and synchrony with different QRS complex duration in complete left bundle branch block (CLBBB) patients with preserved left ventricle ejection fraction (LVEF).Methods A total of 44 patients with CLBBB and LVEF≥50％ were included.All the patients were divided into two groups based on QRS duration,QRS＞150 ms as wide QRS group and 120 ms≤QRS≤150 ms as narrow QRS group.And 30 healthy people were included as control group.Two-dimensional echocardiography and 2D-STE were performed.Ieft ventricle longitudinal peak stain of global,septum and free wall (LS-G,LS-Sept,LS-Lat),standard deviation of time to peak systolic strain for the 18 left ventricular segments (SDt) and index of left yen tricular diastolic function (EDT,E/A and E/e') were measured.Results SDt values of wide QRS group and narrow QRS group were significantly higher than that of control group (both P＜0.01).And SDt of wide QRS group was significantly higher than that of narrow QRS group (P＜0.05).LVEF and LS-G in wide QRS group were significantly lower than those in both narrow QRS group and control group (all P＜ 0.05),while there was no significant difference between narrow QRS group and control group (all P＞0.05).The LS-Sept in wide QRS group and narrow QRS group were both lower than that of control group (both P ＜0.01).And LS-Sept in wide QRS group was lower than that of narrow QRS group (P＜0.01).LS-Lat in narrow QRS group was separately higher than those of both wide QRS group and control group (both P＜0.05),while there was no significant difference of LS-Lat between wide QRS group and control group (P＞0.05).Compared with control group,E/A and EDT decreased and of E/e' increased in both wide QRS group and narrow QRS group (all P＜0.05).While there was no significant difference between wide QRS group and narrow QRS group (all P＞0.05).Conclusion In patients of wide QRS CLBBB with preserved LVEF,left ventricular systolic,diastolic function and synchrony decrease,while left ventricular systolic function of patients with narrow QRS do not significantly decrease.

Objective To investigate the inhibitory effects of silencing expression ofEZH2 gene on the cell proliferation of human QBC939 cells and its mechanisms. Methods The targeting siRNA was designed and trans-fected into QBC939cells. The expressions of EZH2 mRNA and protein were detected by real-time qPCR and west-ern blotting,respectively. The ability of cellproliferationwas analyzed by MTT assay and plate clone formation assay. Cell apoptosis and cycle percentage weremeasured by flow cytometry. Cell senescence was assessed byβ-galactosi-dase dyeing.The expressions of H3K27me3,P14ARF,P16INK4a,P53,P21 and E2F1 proteinwere determined by West-ern blotting.Results Compared with the control group ,the expressions of mRNAand protein were significantly elevat-ed in experimental group. The ability of cellproliferation in the experiment group was significantly down regulated , which could also cause a rise of G1/S phase ,but not a marked variation of apoptosis rate. Silencing EZH2 would induce a obvious senescence phenotype in QBC939 cells. EZH2-siRNA transferredcould also down-regulate the expressions of H3K27me3 and E2F1 protein,while up-regulating the expressions of P14ARF,P16INK4a,P53 and P21 protein in QBC939 cells.Conclusions Silencing EZH2 could induce a significant inhibition on cell proliferation of QBC939 cells,the mechanism of which may be associated with the senescence pathway regulation.

Objective:To develop evaluation index system of Knowledge, Attitude, and Practice Model of esophageal speech training for Otolaryngology nurse, which can provide an initial tool to evaluate the effect of esophageal speech training for Otolaryngology nurse.Methods:Based on the Knowledge, Attitude, and Practice Model and literature research, the initial dimensions and items were developed. The evaluation index system was completed by Delphi technique that 23 experts were invited to evaluate the importance of the dimensions and items in the consulting round. Then the weight value of the index system was computed through analytic hierarchy.Results:For the first and second rounds of the study, respectively, the recovery rates were 78.26% and 100.00%, the expert authority were 0.85 and 0.86, and the Kendall′s W were 0.16 and 0.19 ( P<0.05). Finally, the index system contained 3 dimensions and 30 items. Conclusions:The evaluation index system has the possible scientificity and reliability, and has the feasible thoery and content. In the future, the evaluation index system can be used to evaluate the effect of esophageal speech training for Otolaryngology nurse through the clinical practice improvement.