Objective To study the changes of flow parameters of superior mesenteric artery (SMA) in children with abdominal type Henoch-Schonlein purpura (HSP) using color Doppler ultrasound.Methods Ten children with abdominal type HSP and 17 controls were included in present study.The blood flow parameters of SMA[including peak velocity(PV),end-diastole velocity(EDV),resistant index(RI)]were measured at acute and recovery stage separately.Statistical analysis was conducted among groups.Results PV were (41.57±8.02)cm/s,(33.38±7.44)cm/s and (35.34±9.73)cm/s in acute stage,recovery stage and control group,respectively.There was no statistically significant difference among groups(F=2.471,P=0.10).EDV were(7.63±4.28)cm/s,(4.23±2.57)cm/s and (3.77±0.87) cm/s in acute stage,recovery stage and control group,respectively.There was significantly significant differences between acute stage group and other two groups(t=0.066,P=0.025;t=0.059,P=0.003).RI were (0.85±0.17),(1.00±0.15) and (1.04±0.13) in acute stage,recovery stage and control group,respectively.Also there was significantly significant differences between acute stage group and other two groups(t=1.391,P=0.020;t=1.239,P=0.026).Conclusion For abdominal type HSP in children,the changes of PV,EDV and RI of SMA were significant,which may help us determine the stage of disease.

ObjectiveTo observe the effect of comprehensive therapy on forearm extensor myotenositis.Methods72 cases were divided into two groups: a control group of 36 cases who were given routine treatment,and an experiment group of 36 cases who were given thermotherapy,computerized medium-frequency electrotherapy,physiotherapy,and ADL instruction,etc.After two courses,a simple grading score(for forearms) was used to assess the effect.ResultsOf the control group,22 cases were cured,10 remarkably effective,4 effective;of the experiment,30 cured,4 remarkably effective,2 effective(u=2.04, P<0.05).The difference of average score for forearms before and after the treatment were(6.58±3.17) points for the control and(8.19±3.55) for the experiment(t=2.03,P<0.05).The average days of cure were(5.60±2.54) d for the experiment group,shorter than those for the control(7.00±2.27) d(t=2.05,P<0.05).ConclusionComprehensive therapy is effective on forearm extensor myotenositis.

OBJECTIVETo construct the prokaryotic expression plasmid pET15b-PEP-1-CAT to obtain purified fusion protein of PEP-1-CAT.

METHODSUsing pfu DNA polymerase, the full-length human catalase cDNA was amplified by PCR from pZeoSV2(+)-CAT plasmid, and the PCR product was added with "A" using Taq DNA polymerase. The purified product of CAT cDNA with the base A at its 3' end was ligated with pGEM-T Easy vector and transformed into DH5alpha. The correct recombinant was identified by PCR and Sal I/Bgl II digestion and named as pGEM-T-CAT. Two oligonucleotides were synthesized and annealed to generate a double-stranded oligonucleotide encoding the PEP-1 peptide, which was directly ligated into Nde I/Xho I-digested pET15b. The recombinant plasmid was identified by double-enzyme digestion and named as pET15b-PEP-1. pET15b-PEP-1 and pGEM-T-CAT were further digested by Xho I/BamH I and Sal I/Bgl II, respectively. The purified linear fragment of pET15b-PEP-1 and CAT cDNA fragment were ligated using two pairs of isocaudarners possessing different recognition sequences but producing compatible cohesive ends. The clone with the expected insert was selected using Xho I restriction analysis followed by sequence analysis. The recombinant plasmid was transformed into E. coli BL21(DE3) which was induced by IPTG. The recombinant protein possessed an N-terminal His-tag sequence which could be used to purify the target protein by affinity chromatography on a Ni(2+)-NTA-resin column. The fusion protein PEP-1-CAT was produced and confirmed by specific enzyme activity in vitro.

RESULTSSequence analysis showed that the PEP-1 and the human CAT cDNA sequence of pET15b- PEP-1-CAT had identical sequence with designed PEP-1 peptide and human catalase cDNA sequence in GenBank (accession No. AY028632), respectively. SDS-PAGE and Western blotting confirmed successful expression and purification of PEP-1-CAT fusion protein with specific activity of 77.15 U/g.

CONCLUSIONThe prokaryotic expression plasmid pET15b-PEP-1-CAT has been constructed successfully, and the successful expression and purification of PEP-1-CAT provides a basis for prevention and therapy of various disorders related to oxidative stress.

Base Sequence ; Blotting, Western ; Catalase ; genetics ; metabolism ; Chromatography, Affinity ; Cloning, Molecular ; Cysteamine ; analogs & derivatives ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; Molecular Sequence Data ; Peptides ; genetics ; metabolism ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics ; isolation & purification ; metabolism

OBJECTIVETo investigate the transduction ability of PEP-1-CAT fusion protein into human umbilical vein endothelial cell (HUVECs) and the effects on hydrogen-peroxide (H2O2)-induced oxidative stress injury in these cells.

METHODSWith the use of TA-cloning program and isocaudamer technique, the pET15b-PEP-1-CAT of prokaryotic expression plasmid was successfully constructed. The recombinant plasmid was transformed into E.coli BL21 (DE3) and the protein expression was induced by IPTG. The recombinant protein has an N-terminal His-tag which could be used to purify the target protein by affinity chromatography on a Ni2+-NTA-resin column. The fusion protein PEP-1-CAT was prepared and confirmed by specific enzyme activity in vitro. The purified PEP-1-CAT fusion protein was added on cultured HUVECs in vitro. The transduction ability of PEP-1-CAT fusion protein into cells was analyzed by Western blot and specific enzyme activity. The cells were treated with H2O2 (0.5 mmol/L) alone and in combination with PEP-1-CAT fusion protein for 4 h. Then, the cell viability, lactate dehydrogenase (LDH) and malondialdehyde (MDA) contents were measured.

RESULTSThe PEP-1-CAT fusion protein could be transduced into the cultured HUVECs in a dose- and time-dependent manner and be stable for at least 48 h. After H2O2 administration, cell viability was significantly reduced compared with control group (37.23%+/-5.68% vs. 100%, P<0.05), while LDH leakage (849.3 U/L+/-95.1 U/L) and MDA (8.23 nmol/L+/-1.58 nmol/L) content were significantly higher than that in control group (540.6 U/L+/-65.7 U/L and 2.46 nmol/L+/-1.42 nmol/L, respectively, all P<0.05). Preincubation with PEP-1-CAT proteins at various concentrations (0.25-2 micromol/L) significantly attenuated H2O2-induced cell injury.

CONCLUSIONThe PEP-1-CAT fusion protein could efficiently penetrate HUVECs and the transduced protein could attenuate cellular oxidative stress injury induced by H2O2. The PEP-1-CAT fusion protein might be a new strategy for preventing and treating oxidative stress induced diseases.

Catalase ; metabolism ; Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Endothelial Cells ; metabolism ; Humans ; Hydrogen Peroxide ; Oxidative Stress ; physiology ; Peptides ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo evaluate the androgenic and anti-androgenic effects of GH (growth hormone) transgenic carp in male rats.

METHODSHershberger assay was carried out in castrated male SD rats aged 4-5 weeks. Testosterone propionate (TP) (0.4 mg/kg BW) was administrated for a positive control, GH transgenic carp (3.0 g/kg BW)+TP (0.4 mg/kg BW), parental carp (3.0 g/kg BW) + TP (0.4 mg/kg BW), and flutamide (Flu) (3.0 g/kg BW) were used for negative controls, and vehicle was administered orally for a blank control. All groups were administrated for 10 consecutive days. At the end of the test, animals were anesthetized, then weights of accessory sex organ were measured. Serum testosterone (T), luteinizing hormone (LH), and Follicle-Stimulating Hormone (FSH) levels were detected.

RESULTSThe weights ratios of the accessory sex organs and body weights showed no significant differences between the solvent control and the GH transgenic carp-treated groups. Serum concentrations of FSH, LH, and T of the rats treated with GH transgenic carp + TP showed no significant changes, compared with those treated with TP only.

CONCLUSIONGH transgenic carp does not have any androgenic agonist or antagonist properties in vivo screening tests.

Animals ; Animals, Genetically Modified ; Carps ; genetics ; Follicle Stimulating Hormone ; blood ; Genitalia, Male ; drug effects ; Growth Hormone ; genetics ; metabolism ; pharmacology ; Luteinizing Hormone ; blood ; Male ; Rats ; Testosterone ; blood

OBJECTIVESTo investigate the improvement of specific immune responses induced by plasmid coexpressing hepatitis B surface antigen (HBsAg) and granulocyte-macrophage colony stimulating factor (GM-CSF).

METHODSAll Balb/c (H-2d) mice were immunized with pGM-CSF/S, pS/GM-CSF, pS or control plasmids. 4 weeks later, anti-HBs titer and the levels of IL-2, IL-4 and IFN-gamma in the supernatant of splenocytes were detected using enzyme- linked immunosorbent assay (ELISA), and HBsAg-specific cytotoxic T lymphocytes (CTL) activity was measured with a 51Cr release assay, using P815/S transfectants as target cells.

RESULTSThe anti-HBs antibody titers in the serum, the levels of IL-2 and IFN-gamma, and the CTL activity in pcDNA3.1-GM-CSF-S immuned mice were higher than those in PcDNA3.1-S immunized mice (F=4.176, P<0.01; F=31.188, P<0.01; F=31.796, P<0.01; F

CONCLUSIONIt will improve the specific immune responses induced by HBsAg DNA vaccine after it is binded to the gene of GM-CSF.

Animals ; Enzyme-Linked Immunosorbent Assay ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Vaccines ; immunology ; Interferon-gamma ; biosynthesis ; Interleukin-2 ; biosynthesis ; Interleukin-4 ; biosynthesis ; Lymphocyte Activation ; Male ; Mice ; Mice, Inbred BALB C ; Vaccines, DNA ; immunology

OBJECTIVETo study the suppressive role of emodin on the growth and its effect on the proliferation cycle and apoptotic gene of human lung adenocarcinoma cell line Anip 973.

METHODSThe survival rate and the inhibitory rate of Anip 973 cell in vitro were detected by MTT colorimetric assay and cell growth curve assay at different time points under different concentration of emodin; the cell proliferation cycle and the apoptotic rate were examined with flow cytometry analysis, and Caspase-3 protein expression was measured by immunoblotting assay.

RESULTSEmodin inhibited the proliferation of Anip 973 cell at G0/G1 phase, decreased the cell ratio at S phase and activated the Caspase-3 protein. It suppressed the growth of tumor cells and raised the apoptotic rate in a concentration and time depending manner in a certain extent.

CONCLUSIONEmodin could suppress the proliferation of Anip 973 cell, and its mechanism of anticancer effect may be through activating Caspase-3, to induce apoptosis and block cell cycle.

Adenocarcinoma ; metabolism ; pathology ; Aged ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Blotting, Western ; Caspase 3 ; metabolism ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Emodin ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Male

The aim of study was to explore the effect of mesenchymal stem cells (MSC) transfected with recombinant adenovirus-mediated human vascular endothelium growth factor 165 (ad-vegf-165) on treating ischemic necrosis limbs. Adult SD rats were selected for study. Limb ischemic necrosis model was established by right femoral artery ligation in SD rats. 7 days after ligation, MSC, ad-h-vegf-165-MSC and ad-LacZ-MSC labelled by DAPI were injected into ischemic necrosis limb in rats respectively. One week after injection, the expression of VEGF in ischemic necrosis limbs was detected by Western blot. And at 1, 2 or 4 weeks after injection, the expressions of FVIII and myosin on MSC were evaluated by immunohistochemistry. The results indicated that the MSC labelled by DAPI could be found in the transplantation site of ischemic necrosis limbs under fluorescent microscope. And the number of MSC in MSC-vegf group was more than that in MSC and MSC-LacZ groups. The VEGF expression in MSC-vegf group was higher than that in MSC and MSC-LacZ groups. More importantly, the number of endothelial cells demonstrated characteristic FVIII positive MSC in MSC-vegf group was more than that in MSC and MSC-LacZ group after injections of 1, 2 and 4 weeks. However, the number of myosin positive MSC among MSC-vegf, MSC and MSC-LacZ groups showed no significant difference. It is concluded that MSC transfected with Ad-vegf promotes angiogenesis to repair ischemic necrosis limbs through the increased expression of VEGF.
Adenoviridae ; genetics ; Animals ; Hindlimb ; blood supply ; Humans ; Ischemia ; pathology ; Male ; Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells ; Necrosis ; Neovascularization, Physiologic ; genetics ; Rats ; Rats, Sprague-Dawley ; Transfection ; Vascular Endothelial Growth Factor A ; genetics

To explore the protective effect of Angelica sinensis polysaccharides(ASP) on subacute renal damages induced by D-galactose in mice and its mechanism. Male C57BL/6J mice were randomly divided into 3 groups, with 10 mice in each group. The D-galactose model group was subcutaneously injected with D-galactose (120 mg x kg(-1)), qd x 42; the ASP + D-galactose model group was intraperitoneally injected with ASP since the 8th day of the replication of the D-galactose model, qd x 35; and the normal control group was subcutaneously injected with saline at the same dose and time. On the 2nd day of after the injection, the peripheral blood was collected to measure the content of BUN, Crea, UA, Cys-C; paraffin sections were made to observe the renal histomorphology by HE staining; senescence-associated β-g-alactosidase (SA-β-Gal) stain was used to observe the relative optical density (ROD) in renal tissues; transmission electron microscopy was assayed to observe the renal ultrastructure; the renal tissue homogenate was prepared to measure the content of SOD, GSH-PX, MDA; the content of AGEs and 8-OH-dG were measured by ELISA. According to the result, compared with the D-galactose model group, the ASP + D-galactose model group showed obviously decreases in the content of BUN, Crea, UA, Cysc, AGES, 8-OH-dG, the number of hardening renal corpuscle, renal capsular space and renal tubular lumen, ROD of SA-β-Gal staining positive kidney cells, mesangial cells, basement membrane thickness, podocyte secondary processes fusion and MDA and increases in the number of normal renal corpuscle, ribosome and rough endoplasmic reticulum in podocytes, the activity of SOD and GSH-PX. In Conclusion, A. sinensis polysaccharides can antagonize kidney subacute damages induced by D-galactose in mice. Its protective mechanism may be correlated with the inhibition of the oxidative stress injury.
Angelica sinensis ; chemistry ; Animals ; Deoxyguanosine ; analogs & derivatives ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Galactose ; adverse effects ; Humans ; Kidney ; anatomy & histology ; drug effects ; injuries ; Kidney Diseases ; chemically induced ; drug therapy ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred C57BL ; Oxidative Stress ; drug effects ; Polysaccharides ; administration & dosage ; Protective Agents ; administration & dosage

OBJECTIVETo construct the expression vector pET15b-pep-1-EGFP and purify the fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) for evaluating the cell-penetrating capability of the cell-penetrating peptide PEP-1.

METHODSTwo oligonucleotides encoding PEP-1 was synthesized and annealed to generate PEP-1-encoding DNA. The recombinant plasmid pET15b-pep-1-EGFP was constructed by inserting PEP-1-encoding DNA and enhanced green fluorescent protein (EGFP) cDNA into pET15b. The fusion protein PEP-1-EGFP expressed in E. coli BL21(DE(3)) was purified with Ni(2+)-resin affinity chromatography and transduced into human umbilical vein endothelial cells.

RESULTSSequence analysis confirmed successful construction of the expression vector pET15b-pep-1-EGFP, and the fusion protein PEP-1-EGFP was expressed and purified efficiently with a yield of approximately 14.15 mg/100 ml bacteria medium. SDS-PAGE and Western blotting identified the purified protein as PEP-1-EGFP, and the cell-penetration assay verified that the fusion protein could be transduced into human umbilical vein endothelial cells.

CONCLUSIONThe successful expression and purification of PEP-1-EGFP and its efficient transduction into human umbilical vein endothelial cells provides a basis for PEP-1-mediated biomacromolecular transduction in protein therapy.

Base Sequence ; Blotting, Western ; Cells, Cultured ; Cysteamine ; analogs & derivatives ; metabolism ; Electrophoresis, Polyacrylamide Gel ; Endothelial Cells ; cytology ; metabolism ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Microscopy, Fluorescence ; Molecular Sequence Data ; Peptides ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection ; Umbilical Veins ; cytology