Objective To construct recombinant adenoviral vector expressing autocatalysis caspase- 3 driven by human telomerase reverse transcriptase promoter amplified by two-step transcription amplification (hTERTp-TSTA),and investigate its antitumor effect in ovarian cancer iri vitro and in vivo.Methods Recombinant adenoviruses expressing autocatalytic caspase-3(rev-caspase-3)driven by hTERTp-TSTA were prepared,which were named as AdHTVP2G5-rev-casp3.AdHT-rev-casp3,Ad-rev-casp3 and AdHTVP2G5- EGEP,which express rev-easpase-3 driven by hTERTp,cytomegalovirus promoter(CMVp)and enhanced green fluorescent protein(EGFP),respectively,were used as controls.Western blot,cell counting kit (CCK-8),flow cytometry(FCM)and TdT-mediated dUTP-biotin nick end labeling(TUNEL)were used to detect the expression of p17,active subunit of caspase-3,and p85,and to measure cell survival rates, apoptotic rates and cell cycle distribution in ovarian cell line AO and normal human umbilical vein endothelial cell line HUVEC,following treatments of AdHTVP2G5-rev-casp3.subcutaneous tumor models and abdominally spread tumor models of human ovarian carcinoma using AO cells in BALB/e nude mice were established.Following treatments of AdHTVP2G5-rev-easp3,western blot was used to detect the expression of active caspase-3 in abdominally spread tumors and liver tissues,respectively,and the mouse survival rates and the volume of tumor nodules were measured,and the serum level of alanine transaminase (ALT)and aspartate transaminase(AST)were analyzed to monitor liver damages and HE staining was used to detect the histopathological changes of various organs.Results The levels of p17 expression in AdHTVP2G5-rev-casp3-treated AO cells were significantly higher than that in Ad-rev-casp3 or AdHT-rev- casp3 treated AO cells,while no expression was observed in AdHTVP2G5-rev-casp3-treated HUVEC.There was strong cell killing of AdHTVP2G5-rev-casp3 of hTERT positive AO cells,but not of the hTERT-negative HUVEC cells.Cell survival rate and apoptotic rate of AO cells treated with AdHTVP2G5-rev-casp3 were 17.8% and 40.2%,respectively,significantly different from that treated with AdHT-rev-casp3(75.2% and 16.1%)at the multiplicity of infection(MOI)of 70(P0.05) .Significant expressions of active caspase-3 were shown in AdHTVP2G5-rev-casp3-treated tumors,whereas no expression was shown in liver.In contrast,both tumors and liver tissues showed active caspase-3 expression following treatments of Ad-rev-casp3.AdHTVP2G5-rev-casp3 and Ad-rev-casp3 prolonged mouse survival[mean survival time of(259?14)d and(213?16)d],when compared with treatment with AdHT- rev-casp3[(177?12)d]and AdHTVP2G5-EGFP[(109?7)d;P

Objective To assess the value of MSCT in the evaluation of the anatomy and variation of hepatic veins for living donor liver transplantation (LDLT) donors and the significance of vessel variation in surgical operation. Methods A total of 238 subjects who wanted to be the donors of LDLT underwent MSCT plain and enhanced examination, and the hepatic veins were evaluated. Results Among all 238 subjects, according to Nakamura's classification of hepatic veins, 164 were type Ⅰ, 60 were type Ⅱ, 14 were type Ⅲ. The left hepatic vein (LHV) shared a common trunk with the middle one in 167 subjects. Branches of Ⅷ going along the cross section and diameter larger than 5 mm were detected in 105 subjects. The Ⅳ segment veins drained into MHV in 68, into LHV in 7 subjects. Right inferior hepatic vein with diameter larger than 3 mm was found in 108 subjects, while the distance between RHV and IRHV were larger than 4 cm in 55 subjects. Conclusion MSCT can offer details and exact information about the donors pre-operation, and is an important non-invasive method for the evaluation of hepatic veins of potential LDLT donors.

Objective　To establish animal model for hypertrophic scar and study the characters of its morphology and collagen metabolism. Methods　A total of 64 round wounds (diameter of 6 mm each) with total skin loss were made on the ventral side of rabbit ear using a trephine. Morphology and collagen metabolism of scar wounds were studied at 14,21,35,70 and 98 days after operation, respectively. Results　There were 76% elevated scars developed (45/59 wounds) on the ventral side of rabbit ear at 21 days and 46% elevated scars disappeared (11/24) at 98 days after operation. There were numerous fibroblast proliferation and whorl-arranged collagen fibers at 21 and 35 days. The number of fibroblast decreased, but irregular-arranged fibers still presented in the elevated scars at 70 and 98 days after operation. Hydroxyproline content in elevated scars at 21 days was higher than that in normal skin (P<0.05), and at 35 days was 3 times as that in normal skin and at 98 days was also markedly higher than that in normal skin (P<0.05). Conclusion　Excessive deposition of collagen is a characteristic of hypertrophic scar in rabbits. The conversion of normal scarring to hypertrophic scarring in rabbits occurs at 14～21 days after operation. Both development and regression of hypertrophic scar in rabbit are quicker than that in human.

A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

A three-dimensional (3D) graphic model of a single-chain Fv (scFv) which was derived from an anti-human placental acidic isoferritin (PAF) monoclonal antibody (Mab) was constructed by a homologous protein-predicting computer algorithm on Silicon graphic computer station.The structure, surface static electricity and hydrophobicity of scFv were investigated. Computer graphic modelling indicated that all regions of scFv including the linker, variable regions of the heavy (VH) and light (VL) chains were suitable. The VH region and the VL region were involved in composing the "hydrophobic pocket". The linker was drifted away VH and VL regions. The complementarity determining regions (CDRs) of VH and VL regions surrounded the "hydrophobic pocket". This study provides a theory basis for improving antibody affinity, investigating antibody structure and analyzing the functions of VH and VL regions in antibody activity.

In-vitro assay methods were established to evaluate transactivation and binding activity of compounds on peroxisome proliferator-activated receptor y (PPARγ). Firstly, plasmids were constructed for transactivation assay of PPARγ response element (PPRE) triggered reporter gene expression, and for cell-based binding activity assay of the chimeric receptor, which was fused with PPARγ ligand binding domain (LBD) and yeast transcriptional activator Gal4. Secondly, by using PPARy competitive binding assay based on time resolved-fluorescence resonance energy transfer (TR-FRET), affinities of compounds and drugs to PPARγ were evaluated. In application of these above methods, the PPARγ activating potency and characteristics of different compounds were evaluated, and a novel benzeneselfonamide derivative, ZLJ01, was found to have comparable binding activity and affinity with the well-known PPARy agonist, but lack of PPRE mediated transactivation activity. In preliminary study on in-vitro hypoglycemic activity, ZLJ1 was found to promote insulin-stimulated glucose uptake by liver cells. Therefore, we believe that combining transactivation and binding activity as well as affinity evaluation, the system could be used to screen non-agonist PPARγ ligand as anovel PPARγ modulator
Genes, Reporter ; Hepatocytes ; Hypoglycemic Agents ; chemistry ; Ligands ; PPAR gamma ; agonists ; chemistry ; Plasmids ; Response Elements ; Sulfonamides ; chemistry ; Transcriptional Activation

Objective To investigate the mRNA and protein expression levels of interferon(IFN)-?in the peripheral blood of patients with systemic lupus erythematosus(SLE),to analyze the relationship between IFN-?and disease activity,and to evaluate the role of IFN-?in the pathogenesis of lupus.Methods SYBR green dyeⅠbased real-time quantatives PCR method was used to compare the mRNA expression levels of IFN-?in the peripheral blood leucocyte of SLE patients and healthy controls.Surum levels of IFN-?were measured with ELISA method.Results IFNA1 mRNA expression level in SLE patients(2.8?3.5)was signifi- cantly lower than that of normal controls(12.7?10.7,P=0.000).There was no significant difference between patients treated with glucocorticoid and those without in the expression level of IFNA1(P=0.549).Serum levels of IFN-?in SLE patients was significantly higher than that of normal controls(P=0.003).The SLEDAI score and anti-dsDNA antibody correlated positively,and complement components C3,C4 and leukocytes correlated negatively with serum concentration of IFN-?.IFN-?level correlated with the presence of fever and rash. Conclusion The close relationship between IFN-?serum level and disease activity in SLE patients suggests that IFN-?might be of importance in the disease process.

OBJECTIVETo study the effect of all-trans retinoic acid (ATRA) on U937 cell growth and its mechanism.

METHODSCell cycle was detected by flow cytometry (FCM), expressions of cell cycle associated protein and the p27 related protein were detected by Western blot. The binding of P27 and Skp2 was detected by immunoprecipitation.

RESULTSFCM displayed that ATRA could inhibit the proliferation of U937 cells. At 72 h on 1 micromol/L ATRA treatment, 72% of the cells were arrested at G0/G1 phase. Western blot displayed that ATRA could decrease the expression of cyclin A, up-regulate the expression of p21 and p27, and down-regulate the expression of p27 related proteins Skp2. p27 could bind with Skp2 in U937 cells as detected by immunoprecipitation.

CONCLUSIONATRA may arrest the proliferation of U937 cells through the reduction of Skp2 expression, and finally the induction of the accumulation of p27.

Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Humans ; S-Phase Kinase-Associated Proteins ; metabolism ; Tretinoin ; pharmacology ; U937 Cells

OBJECTIVETo investigate the expression and relationship of p27(kip1) and its nuclear export factor Jab1 during proliferation process of lymphoma cell.

METHODSJurkat and Raji cells were treated with serum starvation and then serum release. The protein and mRNA expression of p27(kip1), Jab1 in the cells were detected by Western blot and RT-PCR respectively. LMB were used for stimulating Jurkat cells during their proliferation process, and then the expression changes of p27(kip1) and Jab1 were detected. An eukaryotic expression plasmid(pcDNA3. 1-myc) containing Jab1 was constructed. Jurkat cell were transfected in vitro with or without pcDNA3. 1-myc-Jab1. Double immunolabelling was used to identify the localization of p27(kip1). Immunoprecipitation was used to detect the combination of p27(kip1) and Jab1.

RESULTSThe growth of Jurkat and Raji cells were blocked by serum starvation. The total protein amount of p27(kip1) increased while that of Jab1 decreased. The reverse changes were happened after serum release, but the mRNA expression of p27(kip1) has no significant change. LMB could inhibit the cell proliferation caused by serum release. The expression of p27(kip1) was up-regulated and Jab1 down-regulated when Jurkat cells were treated with LMB. After pcDNA3. 1-myc-Jab1 infected Jurkat cells for 48 h, the distribution of p27(kip1) was translocated from nucleus into cytoplasma. p27(kip1) and Jab1 could form compound in Jurkat and Raji cells detected by Immunoprecipitation.

CONCLUSIONJab1 may influence the location and expression of p27(kip1) through integrating with p27(kip1), and then participates in regulating the growth of NHL cell through interfering with the function of p27(kip1).

COP9 Signalosome Complex ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; metabolism ; Jurkat Cells ; Peptide Hydrolases ; metabolism ; Plasmids ; RNA, Messenger ; metabolism ; Transfection

Relevant data including diagnosis and treatment model, operation situation, etc. are analyzed in our wound care center and compared with abroad based-on literature retrieval, to find out a suitable way for setting up wound care centers in our country. Wound care center with mode of multidisciplinary cooperation that is "specialist-oriented" was established in our hospital, mirroring the experience of foreign wound care centers. The investigation showed the demands of patients could be met, their psychological and economic pressures as well as social burden might be greatly relieved in our center. For an example, we achieved our purpose readily by setting up a clinical pathway of diabetic foot according to the features of our hospital. The model of multidisciplinary cooperation, with minor adjustments, may be applicable for wound care centers at home and abroad. Our wound care center will establish better system of diagnosis and treatment to conform to the situation in our country.
Burn Units ; organization & administration ; Critical Pathways ; Humans ; Patient Care Team ; Surgicenters ; organization & administration ; Wound Healing