1.Construction of autocatalytic caspase-3 driven by amplified human telomerase reverse transcriptase promoter and its enhanced efficacy of inducing apoptosis in human ovarian carcinoma
Yue SONG ; Keng SHEN ; Chun-Xia HE ;
Chinese Journal of Obstetrics and Gynecology 2000;0(09):-
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
2.MSCT assessment of hepatic veins in living donor liver transplantation donors
Wen SHEN ; Yue CHENG ; Chun XIE ; Ji QI
Chinese Journal of Medical Imaging Technology 2009;25(7):1215-1217
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.
3.Morphological observation and changes of hydroxyproline content in hypertrophic scar of rabbits
Bin SHU ; Linlin HAO ; Zongyao WU ; Xiankai HUANG ; Yue SHEN ; Chun YUAN ; Qimin TANG
Journal of Third Military Medical University 2001;23(3):343-345
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.
4.Construction and Analysis of Three-dimensional Graphic Model of Single-chain Fv Derived from an Anti-human Placental Acidic Isoferritin Monoclonal Antibody by Computer
Chun ZHOU ; Guanxin SHEN ; Huifen ZHU ; Jing YANG ; Yue ZHANG ; Jiannan FENG ; Beifen SHEN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2000;20(1):23-25
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.
5.Construction and Analysis of Three-dimensional Graphic Model of Single-chain Fv Derived from an Anti-human Placental Acidic Isoferritin Monoclonal Antibody by Computer
Chun ZHOU ; Guanxin SHEN ; Huifen ZHU ; Jing YANG ; Yue ZHANG ; Jiannan FENG ; Beifen SHEN
Journal of Huazhong University of Science and Technology (Medical Sciences) 2000;20(1):23-25
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.
6.Establishment and application of screening methods for non-agonist PPARγ ligand.
Yi HUAN ; Jun PENG ; Yue WANG ; Chun-Ming JIA ; Ke WANG ; Ke-Hua WANG ; Zhi-Qiang FENG ; Zhu-Fang SHEN
Acta Pharmaceutica Sinica 2014;49(12):1658-1664
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
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Hepatocytes
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Hypoglycemic Agents
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chemistry
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Ligands
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PPAR gamma
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agonists
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chemistry
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Plasmids
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Response Elements
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Sulfonamides
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chemistry
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Transcriptional Activation
7.The expression of interferon-?in the peripheral blood and correlation with disease activity in systemic lupus erythematosus patients
Shan DENG ; Da-Wei HU ; Yuan WANG ; Nan SHEN ; Yue-Ying GU ; Chun-De BAO ; Shun-Le CHEN ;
Chinese Journal of Rheumatology 2003;0(11):-
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.
8.Expression variation and significance of Skp2 and p27(kip1) during the proliferation of Jurkat cells.
Jian-Xin LU ; Yu-Chan WANG ; Ai-Guo SHEN ; Yue-Ming ZHAO ; Cheng-Long SUN ; Dong-Mei ZHANG ; Chun CHENG
Chinese Journal of Oncology 2008;30(5):330-334
OBJECTIVETo investigate the expression variation and significance of Skp2 and p27(kip1) during the proliferation of lymphoma cell line Jurkat cells.
METHODSThe binding of p27(kip1) and Skp2 in Jurkat cells were detected by immunoprecipitation. Jurkat cells were treated with serum starvation and release synchronization. The expression variation and subcellular localization of p27(kip1) and Skp2 were detected by subcellular fractionation, Western blot and double immunofluorescence labelling.
RESULTSThe results of immunoprecipitation suggested that p27(kip1) and Skp2 could bind each other in Jurkat cells. During the proliferation of Jurkat cells, the protein expression of p27(kip1) decreased and intranuclear p27(kip1) decreased significantly, while the Skp2 protein increased and cytoplasmic Skp2 increased significantly.
CONCLUSIONDuring the proliferation of Jurkat cells, the increased cytoplasmic synthesis of Skp2 may speed up p27(kip1) degradation via the ubiquitin-proteasome pathway, then intranuclear p27(kip1) decreases significantly, leading to an increased cell cycling activity.
Cell Nucleus ; metabolism ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Cytoplasm ; metabolism ; Humans ; Jurkat Cells ; Lymphoma, B-Cell ; metabolism ; pathology ; Protein Binding ; S-Phase Kinase-Associated Proteins ; metabolism
9.Extracorporeal shock wave combined with functional training for the treatment of frozen shoulder.
Kun GAO ; Xi-Chun ZHU ; Li-Hui YUE ; Jing FENG ; Kun SHEN
China Journal of Orthopaedics and Traumatology 2013;26(5):401-403
Adult
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Aged
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Bursitis
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therapy
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Exercise Therapy
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methods
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Female
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High-Energy Shock Waves
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Humans
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Male
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Middle Aged
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Treatment Outcome
10.Morphological observation and changes of hydroxyproline content in hypertrophic scar of rabbits
Bin SHU ; Lin-Lin HAO ; Zong-Yao WU ; Xian-Kai HUANG ; Yue SHEN ; Chun YUAN ; Qi-Min TANG
Journal of Third Military Medical University 2001;23(3):343-345
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.