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Journal of Wuhan university of Technology(Materials science) 2007年第1期22卷 112-116页

作者：刘卫杨祥良 college of Life science and Technology Huazhong University of Science and Technology

To assess the merits of PEGylated poly （lactic-co-glycolic acid）（PEG-PLGA） nanoparticles as drug carriers for tumor necrosis factor-α receptor blocking peptide （TNFR-BP）, PEG-PLGA copolymer, which could be used to prepare the stealth nanoparticles, was synthesized with methoxypolyethyleneglycol, DL-lactide and glycolide. The structure of PEG-PLGA was confirmed with ^1H-NMR and FT-IR spectroscopy, and the molecular weight （MW） was determined by gel permeation chromatography. Fluorescent FITC-TNFR- BP was chosen as model protein and encapsulated within PEG-PLGA nanoparticles using the double emulsion method. Atomic force microscopy and photon correlation spectroscopy were employed to characterize the stealth nanoparticles fabricated for morphology, size with polydispersity index and zeta potential. Encapsulation efficiency （EE） and the release of FITC-TNFR-BP in nanopartieles in vitro were measured by the fluorescence measurement. The stealth nanoparticles were found to have the mean diameter less than 270 nm and zeta potential less than -20 mV. In all nanoparticle formulations, more than 45% of EE were obtained. FITC-TNFR-BP release from the PEG-PLGA nanoparticles exhibited a biphasic pattern, initial burst release and consequently sustained release. The experimental results show that PEG-PLGA nanoparticles possess the potential to develop as drug carriers for controlled release applications of TNFR-BP.

关键词： tumor necrosis factor-α receptor blocking peptide PEG-PLGA stealth nanoparticles ring-opening polymerization controlled and sustained drug release

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Expression and signifi cance of TLR4 and HIF-1α in pancreatic ductal adenocarcinoma

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World Journal of Gastroenterology 2010年第23期16卷 2881-2888页

作者： Jian-Jun Zhang,He-Shui Wu,Lin Wang,Yuan Tian,Jing-Hui Zhang,Hai-Long Wu Department of Pancreatic Surgery,Union Hospital,Tongji medical college,Huazhong university of science and Technology,Wuhan 430022,Hubei Province,china Department of Pediatrics,Union Hospital,Tongji medical college,Huazhong university of science and Technology,Wuhan 430022,Hubei Province,china Laboratory of General Surgery,Union Hospital,Tongji medical college,Huazhong university of science and Technology,Wuhan 430022,Hubei Province,china Department of Pancreatic Surgery Union Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430022 Hubei Province China.

AIM:To investigate the expression of toll-like receptor(TLR) 4,nuclear factor-κB(NF-κB) p65 and hypoxiainducible transcription factor 1α(HIF-1α) in pancreatic ductal adenocarcinoma and their clinical ***:The mRNA of TLR4 and HIF-1α were investigated by real-time polymerase chain reaction in 30 cases of pancreatic ductal adenocarcinoma and its adjacent tissues,and expression of TLR4,NF-κB p65 and HIF-1α protein were detected by immunohistochemistry in 65 cases of pancreatic ductal adenocarcinoma tissues and 38 cases of corresponding adjacent *** relationship between TLR4 or HIF-1α and pathologic features,as well as the association between TLR4 and HIF-1α,were also ***-Meier method was used to assess the impact of expression of TLR4 and HIF-1α on survival of patients with pancreatic ***:The relative quantif ication of TLR4 and HIF-1α mRNA in tumor tissues was 0.81±0.10 and 0.87±0.11,respectively,signif icantly higher than that in adjacent tissues(0.81±0.10 vs 0.70±0.16,P=0.002;0.87±0.11 vs 0.68±0.13,P=0.000).The protein expression of TLR4,NF-κB p65 and HIF-1α in tumor tissues was 69.20%,66.15% and 70.80%,respectively,being signif icantly higher than that in adjacent normal tissues(69.20% vs 39.50%,P=0.003;66.15% vs 31.58%,P=0.001;70.80% vs 36.80%,P=0.001).There was no signif icant correlation between TLR4 or HIF-1α expression and the age,gender,tumor location,the degree of tumor differentiation in the patients(P>0.05).However,there was signif icant correlation between the expression of TLR4 or HIF-1α and tumor size,lymph node metastasis,venous invasion and clinical staging(P<0.05).The expression of TLR4 and HIF-1α had a signif icant impact on survival of patients with pancreatic ***:TLR4,NF-κB p65 and HIF-1α are overexpressed in pancreatic adenocarcinoma,TLR4 may be partly involved in up-regulating HIF-1α,and both synergestically promote development of pancreatic adenocarcinoma.

关键词： Pancreatic ductal adenocarcinoma Toll-like receptor 4 Nuclear factor-κB p65 Hypoxia-inducible factor 1

Purification and Characterization of Two Endo-β-1,4-glucanases from Mollusca,Ampullaria crossean

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Acta Biochimica et Biophysica Sinica 2005年第10期37卷 702-708页

作者： Yan-Hong LI~(1#) Rui GUO~(3#) Qiu-Yu YIN~1 Ming DING~1 Si-Liang ZHANG~3 Gen-Jun XU~(1,2) Fu-Kun ZHAO~(1,2*) 1 Key Laboratory of Proteomics,Institute of Biochemistry and Cell Biology,Shanghai Institutes for Biological sciences.Graduate School of the Chinese Academy of sciences,Chinese Academy of sciences,Shanghai 200031,china 2 college of Life science,Zhejiang Sci-Tech university,Hangzhou 310018,china 3 Bioengineering Institute,East china university of science and Technology,Shanghai 200233,china Key Laboratory of Proteomics Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological Sciences Graduate School of the Chinese Academy of Sciences Chinese Academy of SciencesShanghai 200031 China Bioengineering Institute East China University of Science and TechnologyShanghai 200233 China college of Life science Zhejiang Sci-Tech UniversityHangzhou 310018 China

Two novel endo-β-1,4-glucanases,EG45 and EG27,were isolated from the gastric juice ofmollusca,Ampullaria crossean,by anion exchange,hydrophobic interaction,gel filtration and a secondround of anion exchange *** purified proteins EG45 and EG27 appeared as a single bandon sodium dodecylsulfate polyacrylamide gel electrophoresis with a molecular mass of 45 kDa and 27 kDa,*** optimum pH for CMC activity was 5.5 for EG45 and 4.4-4.8 for *** optimumtemperature range for EG27 was broad,between 50℃ and 60℃;for EG45 it was 50℃.The analysis on thestability of these two endo-β-1,4-glucanases showed that EG27 was acceptably stable at pH 3.0-11.0 evenwhen the incubation time was prolonged to 24 h at 30℃,whereas EG45 remained relatively stable at *** 85% of the activity of EG27 could be retained upon incubation at 60℃ for 24 ***,less than 10% residual activity of EG45 was detected at 50℃.Among different kinds of substrates,bothenzymes showed a high preference for carboxymethyl ***45,in particular,showed a carboxymethylcellulose hydrolytic activity of 146.5 IU/mg *** enzymes showed low activities to xylan (from oatspelt) and Sigmacell 101,and they were inactive to p-nitrophenyl-β-D-cellobioside,salicin and starch.

关键词： cellulase endo-β-1,4-glucanase Ampullaria crossean purification substrate specificity

A　New　Fluorescence　Method　for　the　Measurement　of　Intracellular　Free　Rare　Earth　Ions：　Fura－2　as　an　Indicator

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Chinese Chemical Letters 1996年第9期7卷 820-822页

作者： Pin YANG Tian xin WEI Chun ying Jin xi WANG Da pin FAN(Institute of Molecular science,ShanXi university,Taiyuan,030006)(Chemical and Chemical Industry college of nanjing university,nanjing,210093)(medical college of nanjing university, nanjing, 210093) Institute of Molecular science ShanXi Universíty TaiYuan 030006 Chem. and Chemical Industry college NanJing University NanJing 210093 medical college NanJing University NanJing 210093

Fura-2 was first used to monitor intracellular RE(3+). The characterizations of RE3+-Fura-2 interactions in solutions simulating intracellular ionic composition show that La3+ and Y3+ both form 1:1 RE(3+)-fura-2 complexes. The apparent dissociation constants are 1.61×10(-7)M,and 4.04×10(-7)M,respectively. The mouse myeloma(SP2/0) cells exposed to free RE(3+) or free RE(3+) and some small molecules in body were Studied by single cell canon measurement *** results support the assumption that RE(3+) ions cannot enter the cytoplasm of living cells, and the small molecules used in our experiments couldn't improve the permeability of RE(3+)ions.

关键词： Method the Measurement Intracellular Rare Fluorescence Free

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Orexin A promotes histamine,but not norepinephrine or serotonin,release in frontal cortex of mice

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Acta Pharmacologica Sinica 2005年第2期26卷 155-159页

作者： Zong-yuan HONG~(2.3),Zhi-li HUANG~(3,4,5),Wei-min QU~3,Naomi EGUCHI~3~2School of Life science,university of science and Technology of china,Hefei 230027,china ~3Department of Molecular Behavioral Biology,Osaka Bioscience Institute,Furuedai,Suita,Osaka 565-0874,Japan ~4Department of Pharmacology,Shanghai medical college of Fudan university,Shanghai 200032,china School of Life science University of Science and Technology of China Hefei China Department of Molecular Behavioral Biology Osaka Bioscience Institute Furuedai Suita Japan Department of Pharmacology Shanghai Medical College of Fudan University Shanghai China

Aim:To investigate the effects of orexin A on release of histamine,norepinephrine,and serotonin in the frontal cortex of ***:Samples for measuringhistamine,norepinephrine,and serotonin contents were collected by in vivomicrodialysis of the frontal cortex of anesthetized *** histamine,noradrenaline,and serotonin content in dialysates were measured by ***:Intracrebroventricular injection of orexin A at doses of 12.5,50,and 200 pmol per mouse promoted histamine release from the frontal cortex ina dose-dependent *** the highest dose given,200 pmol,orexin A signifi-cantly induced histamine release,with the maximal magnitude being 230% overthe mean basal *** enhanced histamine release was sustained for 140min,and then gradually returned to the basal ***,no change in nore-pinephrine or serotonin release was observed under application of the same doseof orexin ***:These results suggest that the arousal effect of orexin Ais mainly mediated by histamine,not by norepinephrine or serotonin.

关键词： orexin A histamine noradrenaline serotonin microdialysis frontal cortex

Antioxidant properties of berberine on cultured rabbit corpus cavernosum smooth muscle cells injured by hydrogen peroxide

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Acta Pharmacologica Sinica 2007年第12期28卷 1914-1918页

作者： Yan TAN~(2,3,5),Qiang TANG~4,Ben-rong HU~4,Ji-zhou XIANG~4~2 Institute of Urology,Renmin Hospital,Yunyang medical college,Shiyan 442000 china,~3 Department of Pharmacology,Yunyang medicalcollege,Shiyan 442000,china,~4 Department of Pharmacology,Tongji medical college,Huazhong university of science and Teehnology,Wuhan 430030,china Institute of Urology Renmin Hospital Yunyang Medical College Shiyan China Department of Pharmacology Yunyang Medical College Shiyan China Department of Pharmacology Tongji Medical College Huazhong University of Science and Technology Wuhan China

Aim:To investigate the antioxidant properties of berberine (Ber) on corpuscavenosum smooth muscle cells (CCSMC) in penile erectile ***:We examined the effects of Ber on cultured rabbit CCSMC damaged by hydrogenperoxide (H2O2) through examining cell viability by methyl thiazolyl tetrazoliumassay and assessing the level of malondialdehyde (MDA),superoxide dismutase,(SOD) activity,nitric oxide (NO) products,and lactate dehydrogenase (LDH) re-lease in cells after stimulation with ***:Treatment with 1 mmol/L H2O2significantly decreased the cell viability,NO products,and SOD activity of CCSMCfrom 100% to 48.57%±4.1% (P3 to 6.7±2.1 μmol/L (P30.1±2.6 U/mL (P3 U/L (P3.7±1.3 to 78.4±2.9nmol/mg protein (P<0.01),***,treatment with different con-centrations of Ber (10-1000 μmol/L) inhibited the damaging effects of H2O2,withincreased cell viability (P<0.05 or P<0.01),NO production (P<0.01),and SODactivity (P<0.01) and decreased LDH release and MDA content (both P<0.01).Conclusion:Bet could produce its antioxidant action on oxidative stress-inducedcultured *** effects may be of benefit in the prevention of penileerectile dysfunction.

关键词： berberine antioxidant action corpus cavernosum erectile dysfunction

Regulation of EGF-induced ERK/MAPK Activation and EGFR Internalization by G Protein-coupled Receptor Kinase 2

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Acta Biochimica et Biophysica Sinica 2005年第8期37卷 525-531页

作者： Jingxia GAO~(1,2*) Jiali LI~1 Lan MA~(1*) ~1 Pharmacology Research Center,Shanghai medical college.Fudan university,Shanghai 200032,china ~2 Department of Biochemistry medical college,Tongji university,Shanghai 200092,china Pharmacology Research Center Shanghai Medical College Fudan UniversityShanghai 200032 China Department of Biochemistry Medical College Tongji UniversityShanghai 200092 China

G protein-coupled receptor kinases(GRKs)mediate agonist-induced phosphorylation anddesensitization of various G protein-coupled receptors(GPCRs).We investigate the role of GRK2 on epidermalgrowth factor(EGF)receptor signaling,including EGF-induced extracellular signal-regulated kinase andmitogen-activated protein kinase(ERK/MAPK)activation and EGFR *** immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 *** assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold,1.1-fold and 1.5-fold(P30,60 and 120 min,respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK *** cytometry experimentsshow that GRK2 overexpression has no effect on EGF-induced EGFR internalization,however,it increasesagonist-induced G protein-coupled δ opioid receptor internalization by approximately 40% (P<0.01).Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation,and that themechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhatdifferent at least in receptor internalization.

关键词： G protein-coupled receptor kinase receptor tyrosine kinase epidermal growth factor receptor ERK/MAPK internalization

Adeno-associated virus-mediated bone morphogenetic protein-7 gene transfer induces C2C12 cell differentiation into osteoblast lineage cells

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Acta Pharmacologica Sinica 2005年第8期26卷 963-968页

作者： Min YANG~2, Qing-jun MA~(2,4), Geng-ting DANG~2, Kang-tao MA~3, Ping CHEN~3, Chun-yan ZHOU~(3,4) ~2Department of Orthopedics, Peking university Third Hospital, Beijing 100083, china ~3Department of Biochemistry and Molecular Biology, School of basic medical sciences, Peking university Health science Center, Beijing 100083, china Department of Orthopedics Peking University Third Hospital Beijing China Department of Biochemistry and Molecular Biology School of Basic Medical Sciences Peking University Health Science Center Beijing China

Aim: To investigate the effects of bone morphogenetic protein-7 (BMP7)-expressing recombinant adeno-associated virus (AAV) vector on the differentiation of C2C12 cells. Methods: AAV-BMP7 was packaged by infecting the stable cell clone BHK-21 (integrated with recombinant AAV vector plasmid pSNAV-BMP7) with recombinant herpes simplex virus type 1, which expresses AAV-2 Rep and Cap and possesses AAV packaging functions. Following infection with AAV-BMP7 at multiplicities of infection of lx 10～5 vector genomes per cell and subse-quent culture, C2C12 cells were assessed qualitatively for BMP7 production, alka-line phosphatase activity, osteocalcin production and Cbfal and MyoD expression. Results: C2C12 cells transduced with AAV-BMP7 could produce BMP7 protein until d 28. Alkaline phosphatase in the cultured C2C12 cell lysate was elevated. Secreted osteocalcin in the culture medium was detectable at d 12 and Cbfal mRNA expression level was upregulated, coinciding with downregulation of MyoD in a temporal manner. Conclusion: The present in vitro study demonstrated that AAV-BMP7 could infect and efficiently convert C2C12 cells from myoblasts into osteoblast lineage cells.

关键词： C2C12 adeno-associated virus bone morphogenetic protein-7 gene therapy

Cloning, expression, and characterization of pollen allergens from Humulus scandens (Lour) Merr and Ambrosia artemisiifolia L

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Acta Pharmacologica Sinica 2005年第10期26卷 1225-1232页

作者： Ai-lin TAO~(2,3), Shao-heng HE~(2,4) Allergy and Inflammation Research Institute, Shantou university medical college, Shantou 515031, china," ~The Second Affiliated Hospital of Guangzhou medical college, Guangzhou 510260, china Allergy and Inflammation Research Institute Shantou University Medical College Shantou China The Second Affiliated Hospital of Guangzhou medical college Guangzhou China

Aim: To clone the pollen allergen genes in Humulus scandens (Lour) Merr (LuCao in Chinese) and short ragweed (Ambrosia artemisiifolia L) for recombinant allergen production and immunotherapy. Methods: The allergen genes were selectively amplified in the weed pollen cDNA pool by using a special PCR profile, with the primers designed by a modeling procedure. Following truncated gene cloning and confirmation of the pollen source, unknown 3’cDNA ends were identified by using the 3’-RACE method. The gene function conferred by the full-length coding region was evaluated by a homologue search in the GenBank database. Recombinant proteins expressed in Escherichia coli pET-44 RosettaBlue cells were subsequently characterized by N-terminal end sequencing, IgE binding, and crossreactivity. Results: Three full-length cDNAs were obtained in each weed. Multiple alignment analysis revealed that the deduced amino acid sequences were 83% identical to each other and 56%-90% identical to panallergen profilins from other species. Five recombinant proteins were abundantly expressed in nonfusion forms and were confirmed by using the N-terminal end sequence identity. Sera from patients who were allergic to A artemisiifolia reacted not only with rAmb a 8(D03) derived from A artemisiifolia, but also with recombinant protein rHum s 1(LCM9) derived from H scandens, which confirmed the allergenicity and cross-reactivity of the recombinant proteins from the 2 sources. Comparison of the degenerate primers used for truncated gene cloning with the full-length cDNA demonstrated that alternative nucleotide degeneracy occurred. Conclusion: This study demonstrates a useful method for cloning homologous allergen genes across different species, particularly for little-studied species. The recombinant allergens obtained might be useful for the immunotherapeutic treatment of H scandens and/or A artemisiifolia pollen allergies.

关键词： pollen allergen profilin gene expression

Kinetics of thymocyte developmental process in fetal and neonatal mice

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Cell Research 2003年第4期13卷 265-273页

作者： SHI YUN XIAO, YAN LI, WEI FENG CHENDepartment of Immunology, School of basic medical sciences, Peking university Health science Center, 38 Xueyuan Road, Beijing 100083, china Department of Immunology School of Basic Medical Sciences Peking University Health Science Center Beijing China

Kinetics of thymocyte development in vivo during embryogenesis was pursued. The early development of thymocytes in the fetal and neonatal BALB/c mice was discontinuous, with four waves of cell proliferation occurring at fetal day (Fd) 14 to 17, Fd 18 to day (D) 1 after birth, D 2 to D 5 and D6 thereafter. The first three proliferation waves coincided with the generation of CD4/"CD8/" (DP), TCR+CD4/hiCD8-/lo (CD4 SP), and TCR+CD4-/loCD8int/hi (CD8 SP) thymocytes, respectively. The transition from DN to DP cells was further investigated and it was found out that there were two differential pathways via immature single positive (ISP) cells in the BALB/c mice, each functioning at different fetal ages. One is via TCR-CD4-CD8+ cells, occurring between Fd 15 and Fd 17 and the other is via TCR-CD4+CD8- cells, occurring from Fd 17 until birth. In contrast, the TCR-CD4-CD8+ pathway dominated overwhelmingly in the C57BL/6 mice. These findings shed new light on the hypothesis that the differential pathway preferenc

关键词： fetal thymus neonatal mice thymocyte ontogeny.