Objective To design an outpatient appointment system based on ASP.NET which gains advantages over the existing system in database stability,functional module integrity and interface friendliness.Methods The ASP.NET-based system was developed with Microsoft Visual Studio 2010 as the platform,Microsoft SQL Server 2005 to create data and kinds of components to establish database and application interface.Results The system enhanced hospital registration and patient satisfaction.Conclusion The system is worthy promoting for outpatient appointment in the hospital.

OBJECTIVETo explore the method that use combined pectoralis major muscle flap and intermediate split thickness skin graft to reconstruct giant cervical pharyngeal fistula.

METHODSUse pectoralis major muscle flap combined with intermediate split thickness skin graft to reconstruct giant cervical pharyngeal fistula caused by malignant tumor surgical treatment and radiotherapy.

RESULTSIn this group, 11 flaps survived after operation, while 2 of them got delayed union after proper treatment. The following -up showed good function and shape, all the flaps remain alive. The patients' swallowing function were recovered and got good contours.

CONCLUSIONSPectoralis major muscle flap has enough volume and vascular pedicle length. Intermediate split thickness skin is easy to survive and has stable colour. Through this combined reconstructive methods, we got good clinical results to treat giant cervical pharyngeal fistula.

Adult ; Aged ; Cutaneous Fistula ; surgery ; Female ; Humans ; Male ; Middle Aged ; Pectoralis Muscles ; transplantation ; Pharyngeal Diseases ; surgery ; Reconstructive Surgical Procedures ; methods ; Surgical Flaps

OBJECTIVETo label human insulin-like growth factor-I (hIGF-I) eukaryotic expression vector with green fluorescent protein (GFP) for the repair of articular cartilage defects.

METHODSGFP cDNA was inserted into pcDNA(3.1)-hIGF-1 to construct the co-expression vector with two multiple cloning sites mammalian expression vector under two cytomegalovirus promoters/enhancers respectively. Recombinant pcGI was transfected into NIH 3T3 cells with the help of lipofectamine.

RESULTSEnzyme digestion and agarose gel electrophoresis analysis revealed that pcGI vector contained correct GFP and hIGF-I cDNA. Expression of hIGF-1 and GFP was confirmed in transfected NIH 3T3 cells by immunocytochemical analysis and fluorescence microscopy.

CONCLUSIONShIGF-I eukaryotic expression vector has been successfully labeled with GFP.

Cartilage, Articular ; injuries ; Cells, Cultured ; Eukaryotic Cells ; cytology ; physiology ; Fibroblasts ; cytology ; physiology ; Gene Expression Regulation ; Genetic Therapy ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; pharmacology ; Humans ; Immunohistochemistry ; Insulin-Like Growth Factor I ; genetics ; therapeutic use ; Luminescent Agents ; pharmacology ; Sensitivity and Specificity ; Transfection ; methods ; Wounds and Injuries ; diagnosis ; therapy

OBJECTIVETo label the primary articular chondrocytes overexpressing human insulin-like growth factor (hIGF 1) with green fluorescent protein (GFP) for repair of articular cartilage defects in rabbits.

METHODSGFP cDNA was inserted into pcDNA3.1 hIGF 1 to label the expression vector. The recombinant vector, pcGI, a mammalian expression vector with multiple cloning sites under two respective cytomegalovirus promoters/enhancers, was transfected into the primary articular chondrocytes with the help of lipofectamine. After the positive cell clones were selected by G418, G418-resistant chondrocytes were cultured in medium for 4 weeks. The stable expression of hIGF 1 in the articular chondrocytes was determined by in situ hybridization and immunocytochemical analysis and the GFP was confirmed under a fluorescence microscope. Methyl thiazolyl tetrazolium (MTT) and flow cytometer methods were employed to determine the effect of transfection on proliferation of chondrocytes. Gray value was used to analyze quantitatively the expression of type II collagen.

RESULTSThe expression of hIGF 1 and GFP was confirmed in transfected chondrocytes by in situ hybridization, immunocytochemical analysis and fluorescence microscope observation. Green articular chondrocytes overexpressing hIGF 1 could expand and maintain their chondrogenic phenotypes for more than 4 weeks. After the transfection of IGF 1, the proliferation of chondrocytes was enhanced and the chondrocytes could effectively maintain the expression of type II collagen.

CONCLUSIONSThe hIGF 1 eukaryotic expression vector containing GFP marker gene has been successfully constructed. GFP, which can be visualized in real time and in situ, is stably expressed in articular chondrocytes overexpressing hIGF 1. The labeled articular chondrocytes overexpressing hIGF 1 can be applied in cell-mediated gene therapy as well as for other biomedical purposes of transgenic chondrocytes.

Animals ; Cartilage, Articular ; metabolism ; Cells, Cultured ; Chondrocytes ; metabolism ; Flow Cytometry ; Genetic Markers ; Genetic Therapy ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Insulin-Like Growth Factor I ; genetics ; metabolism ; Luminescent Agents ; RNA, Messenger ; analysis ; Rabbits

OBJECTIVETo investigate how F261S mutation identified from Chinese obese patients affects the function of melanocortin 4 receptor (MC4R) and to analyze the obesity-related phenotypes in subjects carrying the F261S mutation.

METHODSF261S mutant of MC4R was generated by site-directed mutagenesis. Plasmids encoding wild-type or F261S mutant of MC4R were transfected into HEK293 and COS-7 cells to examine their functional characteristics. Signaling properties of F261S MC4R were assessed by measuring intracellular cAMP levels in response to alpha-MSH stimulation. Cell surface expression of F261S MC4R was compared with that of wild-type MC4R. Clinical examinations were performed in subjects carrying F261S mutation and in non-mutated controls.

RESULTSThe alpha-MSH-stimulated reporter gene activity was significantly reduced in cells expressing F261S MC4R, with a maximal response equal to 57% of wild-type MC4R. The F261S mutation also led to a significant change in the Es50 value compared with the wild-type receptor (P<0.01). Immunofluorescent assay revealed a marked reduction in plasma membrane localization of the MC4R in cells expressing the F261S mutant receptor. The resting metabolic rate and fat composition of the mutant carriers were not significantly different from those of the non-mutated obese controls.

CONCLUSIONSThe decreased response to alpha-MSH due to the intracellular retention of MC4R may cause early-onset obesity in the F261S pedigree of Chinese.

Adult ; Age of Onset ; Aged ; Animals ; COS Cells ; Cercopithecus aethiops ; Child ; China ; Female ; Humans ; Male ; Middle Aged ; Mutation ; Obesity ; epidemiology ; metabolism ; Pedigree ; Receptor, Melanocortin, Type 4 ; genetics ; metabolism

OBJECTIVEAlthough HIV-1 infection is prevalent in many regions in China, it remains largely unknown on the biological characteristics of dominant circulating isolates. This study was designed to isolate the circulating viral strains from different prevalent regions and to characterize their biological properties and neutralization sensitivity.

METHODSPrimary viruses were isolated from fresh PBMCs using the traditional co-culture method and their capacity of inducing syncytium was tested in MT-2 cells. Meanwhile, their coreceptor usage was determined with two cell lines: Magi and GHOST (3) stably expressing CD4 and the chemokine receptor CCR5 or CXCR4. Furthermore, the sensitivity of these viruses to neutralization by HIV-1-infected patients' plasma which were highly active to neutralize SF33 strain, was quantified in GHOST cell-based neutralization assay.

RESULTSSix primary viral strains were isolated from 4 separated regions. Isolates LTG0213, LTG0214 and XVS032691 induced syncytia in MT-2 cells, and used CXCR4 as coreceptor. Isolates XJN0021, XJN0091, or SHXDC0041 did not induce syncytia, and used CCR5 as coreceptor. Overall neutralization sensitivity differed among four representative strains: HIV-1 XVS032691 > LTG0214 >XJN0091 approximately SHXDC0041.

CONCLUSIONThe neutralization sensitivity of HIV isolates is linked with the phenotype of isolates, in which syncytium-inducing (SI) or CXCR4-tropic (X4) viruses are more easily neutralized than non-syncytium-inducing (NSI) or CCR5-tropic (R5) viruses. The genetic subtypes based on the phylogeny of env sequences are not classical neutralization serotypes.

CD4-Positive T-Lymphocytes ; metabolism ; Cell Line ; Cells, Cultured ; Chemokines ; genetics ; immunology ; China ; Coculture Techniques ; methods ; Giant Cells ; ultrastructure ; virology ; HIV Infections ; virology ; HIV Seropositivity ; genetics ; immunology ; HIV-1 ; immunology ; isolation & purification ; physiology ; Humans ; Neutralization Tests ; Receptors, CCR5 ; metabolism ; Receptors, CXCR4 ; metabolism ; Virus Replication

OBJECTIVETo investigate the relationship between resting energy expenditure (REE) and patterns of obesity/regional fat parameters in Chinese adults.

METHODSBody mass index (BMI), fat mass (FM), fat-free mass (FFM) were assessed in 109 Chinese adults (52 men and 57 women), and their abdominal visceral adipose tissue area (VA) and subcutaneous fat area (SA) were measured using magnetic resonance imaging (MRI) measurements. REE was measured with indirect calorimetry and compared with normal and obese subjects. Multivariate analysis was used to study the factors related to REE.

RESULTSThe resting energy expenditure per kilogram of body weight (REE/kg) was closely related with the area of abdominal visceral fat measured with MRI. REE/kg was significantly lower in overweight/obesity subjects than in normal-weighted subjects, and significantly lower in subjects with abdominal obesity (VA > or = 100 cm2) than in subjects with non-abdominal obesity (VA < 100 cm2, BMI > or = 25 kg/m2). In the stepwise regression analysis of REE/kg on regional fat parameters, VA in men and women and SA in women were independent factors reversely related to REE/kg.

CONCLUSIONREE/kg is associated with the visceral fat area and more prominent in men. REE/kg can be used as an index in the pathophysiology of intra-abdominal obesity.

Adipose Tissue ; metabolism ; Body Composition ; Body Mass Index ; Body Weight ; Energy Metabolism ; Fats ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Obesity ; metabolism ; Rest ; physiology ; Viscera ; metabolism

To explore the genetic characteristics of viral quasispecies in HIV-1 CRF07_BC infections among intravenous drug users (IDU), the gp120 fragments of HIV-1 env gene were amplified from plasma samples collected from 6 CRF07_BC infected persons using single genome amplification and sequencing (SGA/ SGS) method, and 11 to 28 sequences were obtained from these samples, respectively, A neighbor-joining phylogenetic tree was reconstructed to describe the genetic characteristics of viral quasispecies. The Simplot, segments' phylogenetic trees and diversity plots based on average pairwise distance (APD) were used to identify the recombination events between quasispecies. The SGA sequences derived from single specimen formed a large monophyletic cluster in the neighbor-joining phylogenetic tree and showed the complex topologic structures of viral quasispecies. Of the 6 CRF07_BC infected patients, only one possessed the high genetic homogeneity, whereas the other five individuals showed high heterogeneity, with two to four subclusters inside the monophyletic cluster for each specimen. In addition, the recombinant events were identified among viral quasispecies from 3 cases. The results show SGA technique and phylogenetic analyses are useful tool to investigate the intrahost CRF07_BC gp120 complex quasispecies variation and high genetic diversity.
Adult ; Drug Users ; Female ; HIV Infections ; virology ; HIV-1 ; classification ; genetics ; isolation & purification ; Humans ; Male ; Molecular Sequence Data ; Phylogeny ; Substance Abuse, Intravenous ; virology ; Young Adult

OBJECTIVE:To prepare and characterize Fluorescent dye 1,1′-octacosyl-3,3,3′,3′-tetramethylindocarbocyanine iodide(DiR)-loading polyethylene glycol-poly lactic-co-glycolic acid(DiR-PEG-PLGA)nanocapsules,and to evaluate its biocompatibility in vitro. METHODS:Using PLGA and PEG-PLGA as carrier,DiR-PEG-PLGA nanocapsules were prepared by modified ultrasonic emulsification method. The particle size,Zeta potential,morphology,stability and fluorescence in vitro of nanocapsules were detected respectively. MTT assay was used to evaluate cytotoxicity in vitro of nanocapsules to human-derived HL7702 hepatocytes,and hemolysis test was carried out to investigate its hemolysis effects. RESULTS:Prepared DiR-PEG-PLGA nanocapsules were spherical with a clear core-shell structure. The average particle size was(507.53 ± 7.87)nm,polydispersity coetficient of particle size was 0.306 1±0.001 5 and Zeta potential was(-35.20±0.92)mV with good stability within 6 months under 4℃. Fluorescence signal intensity(y)of nanocapsules was increased linearly with DiR mass concentration(x)in vitro. The linear eguation was y=0.345 2x+0.433 4(R2=0.997 3).The toxicity of nanocapsules to HL7702 cells was between 0-1 degree,and no hemolytic effect was observed. CONCLUSIONS:The study successfully prepare fluorescent DiR-PEG-PLGA nanocapsules with high biocompatibility in vitro,which is further expected to become a safe optical drug carrier.

Objective Antibody drugs are one of the hot topics in biomedical research.This study aims to develop egg yolk antibodies (IgYs) against human isomaltase and determine their biological activities.Methods The purified recombinant isomaltase protein was used as an antigen to immunize egg-laying hens in combination with complete Freund adjuvant (CFA).Anti-isomahase IgYs were extracted by water dilution-sodium sulfate extraction assay and further analyzed for their purity,specificity,titer and stability by SDS-PAGE,Western blot and ELISA respectively,and their inhibitory effect on human alpha-glycosidase enzymes was evaluated by the PNPG method.Results Anti-isomaltase IgYs were obtained,with a titer of 1 ∶ 12800,capable of specifically binding human isomaltase,and with a good thermal stability,acid/alkali stability and pepsin resistance.Conclusion Anti-human isomaltase IgYs were successfully prepared,which may provide an experimental ground for further investigation of oral antihyperglycemic agents for type Ⅱ diabetes mellitus.