BACKGROUND:Preterm premature rupture of the membrane is a frequent complication during gestational period. Three-dimensional culture can help damaged tissue restore anatomical integrality,being worth for clinical application. OBJECTIVE:To explore biological characteristics of human amniotic cells three-dimensionally cultured by fibrin scaffold,study the possibility of cell/scaffold composite material for repairing preterm premature rupture of the membrane,and to compare with normal monolayer culture. DESIGN,TIME AND SETTING:An in vitro cell/scaffold study was performed at the Basic Institute of Chongqing University of Medical Science between December 2007 and November 2008. MATERIALS:Amnion tissue derived from uterine-incision delivery women was provided by the Department of Obstetrics and Gynecology,the First Affiliated Hospital,Chongqing University of Medical Sciences;fibrinogen was provided by Sigma,USA. METHODS:Either epithelial cells or mesenchymal cells,being isolated from human amniotic membrane,were cultivated using enzyme digestion and repeated adherence methods. Fibrinogen was conglomerated,and epithelial cells of logarithmic growth were plated onto the surface of fibrin scaffold to simulate a three-dimensional culture in vitro. Before conglomerating fibrinogen,mesenchymal cells of logarithmic growth were mixed with fibrin solution to simulate a three-dimensional culture in vivo. In addition,either epithelial cells or mesenchymal cells were incubated onto 24-well plate for normal monolayer culture. MAIN OUTCOME MEASURES:Cell morphology using inverted microscopy,proliferation of mesenchymal cells,and interaction between mesenchymal cells and scaffold under different culture environments. RESULTS:Epithelial cells were round and smooth on the surface of fibrin scaffold,pseudopodia were stretched out,and microvillus were rich. Mesenchymal cells in fibrin glue were fusiform in shape and stretched out along scaffold. Cells formed net structure at different surfaces. By three-dimensional culture,proliferation of mesenchymal cells was stable but slow compared to monolayer culture. By in vivo three-dimensional culture,fibrin glue gradually shrank and gel thickness gradually decreased. On the 5th day,the gel thickness was only 40% for the initial thickness,and on the 15th day,gel thickness was 10%. CONCLUSION:Both epithelial cells and mesenchymal cells can sterically grow on fibrin scaffold,and the proliferation is stable. Fibrin glue gradually shrinks in cell/fibrin composite scaffold,suggesting that amniotic cells/fibrin composite scaffold can simulate body tissues to repair preterm premature rupture of the membrane.

Objective To investigate the ex tr action technique for seperating the active components in the root of Salvia mi ltiorrhizae bunge by supercritical fluid, and to analyze the extracted product s by HPLC-MS n . Methods The extraction condition s were established as follows: 950ml?L -1ethanol as the first entrainer, t he pressure of 20.0 MPa, temperature at 45 ℃, and extracting time 1 h; then 100 mL?L -1 ethanol was selected as the second entrainer, pressur e was 30.0 MPa, temperature was 65 ℃, and extracting time was 3 h. Results Compared with traditional refluxing extraction and ultrasonic extraction, supercritical fluid extraction was better and more effect ive. Conclusion Supercritical extraction is simple, highly selec tive and efficient in extracting the active components in Salvia miltiorrhizae bunge.

Objective To investigate the role of erythrocyte complement receptor type 1 (CR1) in the pathogenesis of chronic urticaria.Methods Venous blood samples were collected from 59 patients with chronic urticaria (including 14 cases of dermatographism and 45 chronic idiopathic urticaria) and 29 healthy human controls.Flow cytometry was carried out to quantify the expression level of CR1,and double-antibody sandwich enzyme-linked immunosorbent assay to determine the serum level of immunoglobulin E (IgE),complement C3,C4 and 50％ complement hemolytic activity (CH50).Differences in these parameters were analyzed by one-way ANOVA and independent samples t-test,and correlation between these paramenters by Pearson correlation analysis.Results The expression level (expressed as mean fluorescence intensity per 10 000 erythrocytes) of CR1 was significantly higher in patients with dermatographism and chronic idiopathic urticaria than in the healthy controls (35.06 ± 2.06 and 29.17 ± 1.53 vs.20.46 ± 2.57,t =4.20 and 3.33,both P ＜ 0.05),while no statistical difference was observed between the patients with dermatographism and chronic idiopathic urticaria (P ＞ 0.05).Increased total serum IgE levels were observed in patients with dermatographism and chronic idiopathic urticaria compared with the healthy controls ((769.89 ± 123.0) μg/L and (340.09 ± 29.74) μg/L vs.(107.63 ± 88.79) μg/L,t =5.58,5.85,both P ＜ 0.05),and in patients with dermatographism compared with those with chronic idiopathic urticaria (t =3.49,P ＜ 0.05).For patients with chronic urticaria,there was a statistical difference in the expression level of CR1 between individuals (n=22) with total serum IgE levels ranging from 0 to 240 μg/L and those (n =17) higher than 500 μg/L (24.45 ± 10.83 vs.33.09 ± 11.86,t =3.33,P＜ 0.05).The total serum IgE levels were positively correlated with the level of CR1 (r =0.27,P ＜ 0.05),but uncorrelated with that of complement C3 (r =0.16,P ＞ 0.05) or C4 (r =-0.08,P＞ 0.05).The level of complement C3 was positively correlated with that of C4 (r =0.54,P ＜ 0.01).One-way ANOVA revealed no significant difference in the serum levels of complement C3,C4,or CH50 between the patients with dermatographism,patients with chronic idiopathic urticaria and healthy controls (all P ＞ 0.05).Conclusion CR1 is abnormally expressed in patients with chronic urticaria.

Objective To explore the predictive value for coronary heart disease by epicardial adipose tissue(EAT) thickness and carotid intima-media thickness(IMT) measured with high-frequency ultrasound.Methods According to the results of coronary angiography, the cases were divided into the normal control group (29 patients),coronary artery disease group with single-vessel lesion (43 patients),coronary artery disease group with multi-vessels lesion (28 patients), respectively. EAT and IMT were measured by high-frequency ultrasound. Results The EAT in the three groups were (4.8 ± 1.3) mm, (7.6 ± 1.8) mm,(10.1±2.6) mm respectively, and the IMT were (0.8±0.1)mm,(1.0±0.2)mm,(1.1 ± 0.2)mmrespectively. In either coronary artery disease group with single-vessel lesion or multi-vessels lesion, the EAT and IMT were significantly higher than those in the normal control group ( P＜ 0.01 ). And the difference between groups of single-vessel lesion and multi-vessels lesion was also statistically significant (P＜ 0.01). The areas under receive operating characteristic(ROC) curve to predict coronary heart disease by EAT and IMT was 0.947 and 0.917, respectively, there was no significant difference between the two areas. For patient with coronary artery stenosis＞50%, the sensitivity and specificity of EAT＞6 mm were 90.1% and 86.2% ,respectively,the sensitivity and specificity of IMT＞0.85 mm were 87.3% and 82.8%,respectively. Conclusions EAT and IMT measured by high-frequency ultrasound can precisely predictcoronary heart disease. EAT can be a new predictor to diagnose coronary heart disease.

Objective To investigate the influence of injection color regents concentration on completed resection rate, operation time and injection volume of patients with intestinal tumor by ESD.Methods 90 elderly patients with intestinal tumor by ESD were chosen from June 2013 to June 2016. They were randomly divided into 3 groups including A group (30 patients) with glycerin fructose used alone, B group (30 patients) with glycerin fructose combined with 2% mythylene blue on the basis of glycerin fructose and C group (30 patients) with glycerin fructose combined with 4% mythylene blue on the basis of glycerin fructose; and the completed resection rate of tumor, operation time, injection volume and complication incidence in the peri-operative period of 3 groups were compared.Results There was no signiifcant difference in the completed resection rate of tumor among the 3 groups (P < 0.05). The operation time and injection volume of C group were signiifcant better than A group and B group (P< 0.05). There was no signiifcant difference in operation time and injection volume between A group and B group (P < 0.05). There was no signiifcant difference in the complication incidence in the peri-operative period among the 3 groups (P < 0.05).Conclusion Compared with glycerin fructose used alone and glycerin fructose combined with 1% mythylene blue for 2 mg, glycerin fructose combined with 1% mythylene blue for 4 mg by intestinal submucosal injection on patients with intestinal tumor by ESD can efifciently shorten the operation time and reduce the injection volume.

Objective To explore the spiral the normal representations of permanent teeth and alveolar bone of multi-slice spiral(MSCT). Methods 30 adults with normal teeth were studied by multislice spiral CT scanner,window width and window level was 3000~5000 HU and 1200~1500 HU respectively.MSCT findings were analysed using multiplanar reformation,dental panorama and 3D reconstruction were done in all cases.Results The tooth were showed as multilaryer structures with different density at MSCT because of the mineral content of different part of tooth,from high to low density was enamel,circumpulpal dentin,outside dentin,cementum and pulp.The periodontal membrane was low density located beween the cementum and lamina dura and the thickness was homogeneous.The bone cortex of alveolar bone was successive,the bone trabecula appeared as wirelike or reticulate.Conclusion The structures of permanent teeth and alveolar bone can be displayed clearly by MSCT.Multiplanar reformation,dental panorama and 3D reconstruction are helpful to display the structures of teeth and alveolar bone as well as the relationship around structures.

According to the codon bias of Pichia pastoris, the mature insect neurotoxin gene LqhIT2 was synthesized based on its amino acid sequence and was cloned to vector of PET-30a (+) and pPIC9K respectively. The fusion protein expressed in Escherichia. coli was induced with IPTG and purified with Ni-NTA His Bind Column. The purified fusion protein was used to immunize BALB/c mice, and antiserum obtained was highly specific with the titer of over 1:128 000. Using the antiserum, high-level expression transformants of P. pastoris were screened by dot blotting. The highest expression of recombinant LqhIT2 was about 9 mg/L in baffled flasks. The fusion protein of LqhIT2 expressed in E. coli was not toxic to locust, but the recombinant LqhIT2 expressed in P. pastoris had insecticidal activity against locust through injection.
Animals ; Escherichia coli ; genetics ; metabolism ; Immune Sera ; biosynthesis ; Immunization ; Mice ; Mice, Inbred BALB C ; Neurotoxins ; biosynthesis ; genetics ; immunology ; Pichia ; genetics ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Scorpion Venoms ; biosynthesis ; genetics ; immunology

Objective To investigate the relatio nship between the DNA methylation status of gluco-corticoid receptor (GR) gene promoter and mRNA expression level of GRα gene of peripheral blood mononuclear cells (PBMCs) in patients with systemic lupus erythematosus (SLE). Methods Fifteen new onset SLE patients and fifteen healthy controls were enrolled in this study. The DNA methylation status of GR gene promoter 1 of PBMCs was detected through bisulfite sequencing polymerase chain reaction (PCR). The mRNA expression of GRα, DNA methyltransferases, growth arrest and DNA damage-induced 45α (GADD45α) of PBMCs was detected using the quantitative real-time polymerase chain reaction method. T-test and χ2-test were used to detect the differences between the two groups, Pearson's correlation coefficient was used to analyze the linear correlation between two variables. Results Compared with healthy controls, the mRNA expression of GRα was signi-ficantly declined in SLE patients (10±5, 17±7, t=2.69, P<0.05), and the mRNA expression of DNMT1 and GADD45α was significantly elevated in SLE patients (t=3.11, P<0.05 and t=2.98, P<0.05). The overall mean methylation status of the 142 CpG islands of the four promoters was significantly elevated in SLE patients [(16±8)%vs (11±6)%, t=2.75, P<0.05]. The global methylation status of PBMCs in SLE patients was obviously lower than healthy controls (t=4.39, P<0.05). Conclusion Hypermethylation of GRα promoter may result in GRαgene low expression in PBMCs of patients with SLE.

Objective:To elucidate the characterization of CD8+T cell in H1N1 influenza vaccine for children.Methods:PBMCs were isolated from 31 children aged from 3 to 6 years old who had accepted H 1N1 influenza vaccine during December 2009 to January 2010.The lymphocytes were joined with the H 1N1 influenza vaccine as experimental group and cultured .The experiment set without vaccine group as control group .At last we detected the surface molecules by FCM .The CCK-8 assay was added to detecting cellular proliferation and cellular proliferation index were detected by CCK-8.Results: CD8+T cells of PBMC in the two groups were 13.41%and 9.41%,P>0.05.CD8+CD45RAA+naive T cells in the two groups were up to more than 80%,P>0.05.The proportion of CD8+CD45ROA+memory T cells in two groups were up to 17%-19%,P>0.05.Two subsets of CD8+CD45ROA+memory T cells :CCR7+and CD62L+single positive memory T cell subsets in the experimental group were significantly lower than that of the control group,P<0.05.The CCK-8 assay was added to detect cellular proliferation .Only 51.16% of which cellular proliferation index was greater than 0.8,with none was greater than 1 in this study.Conclusion:This study showed that the CD4+T cells were low-level,naive T cells (CD8+CD45RAA+)were higher,with antigen stimulation and response.H1N1 vaccination specific memory T cells were few in number , specific memory T cell subsets were diversity , control memory cells were the main phenotypic characteristics .Cellular proliferation index showed that the proliferation of specific CD 8+T cells vaccine was poor .

Objective To explore the correlation between epicardial adipose tissue(EAT) thickness and cardiovascular risk factors in patients with coronary artery disease.Methods According to the results of coronary angiography,84 cases of patients undergone coronary angiography were divided into the normal control group(28 patients),coronary artery disease group with single-vessel lesion (28 patients),coronary artery disease group with multi-vessels lesion(28 patients),respectively,then measured the EAT thickness and carotid intima-media thickness(IMT) by high-frequency ultrasound.A correlation analysis was carried out between the EAT and IMT and cardiovascular risk factors.Results In either coronary artery disease group with single-vessel lesion or multi-vessels lesion,the thicknesses of EAT and IMT were significantly higher than those in the normal control group(P ＜0.01),and the difference between group of single-vessel lesion and multi-vessels lesion was also statistically significant (P ＜0.01).A correlation analysis between the EAT and IMT and cardiovascular risk factors showed that there were significantly positive correlations between EAT and the IMT,age,weight,waist circumference,body mass index,low-density lipoprotein,Creactive protein (r = 0.124～0.790,P ＜0.05 or P ＜0.01),and the significantly negative correlation between EAT and high-density lipoprotein (r = - 0.203,P ＜ 0.05).Correlation between EAT and the IMT was 0.678,0.713,0.737 in the normal control group,group with single-vessel lesion,group with multivessels lesion,respectively.Conclusions There were favorable correlation between EAT and carotid IMT in cardiovascular risk factors.The more severity of coronary heart disease,the more high correlations of EAT and IMT.EAT can be a valuable index in evaluating coronary artery disease.