Objectives To express human TFPI-2 gene in E.coli and get the recombinant protein;to investigate the recombinant activities of antiplasmin and antitumor.Methods (1) Encoding region of mature protein of human TFPI-2 was amplified by PCR.The 645 bp PCR product was cloned into expression vector pET28a and transformed into BL21 strain to get expression.(2)Identifying the DNA segment of TFPI-2 with enzyme digestion and colony PCR and the TFPI-2 fusion protein with western blot respectively.(3)To establish the kinetics assay for detecting the TFPI-2 and test it′s inhibiting plasmin activities.(4)To assess ovarian tumor cell migratory and invasive behaviors with the Boyden chamber in vitro.Results (1)Expression plasmid of recombinant TFPI-2 was constructed and high-level expression of TFPI-2 was produced as fusion protein. (2) The TFPI-2 fusion protein was identified by western blot. (3) Antiplasmin activity of the TFPI-2 was confirmed in the fluid and on the cell surface. (4) In invasion assay, the number of the A2780 and A2780-TFPI-2 groups transferred the Matriged matrix-coated PVPF membrane was obvious decreased compared with that of A2780 group(P0.05).Conclusion (1) Prokaryotic expression of TFPI-2 gene was got, which can provide the rich experimental material for investigating the role of TFPI-2 in relative fields; (2)Recombinant TFPI-2 has antiplasmin activity, which provides the experiment basis for investigating the role of recombinant TFPI-2 in human ovarian tumor migration and invasion in vitro; (3) The recombinant TFPI-2 inhibits the invasive ability of human ovarian tumor cells in vitro, but has no effect on the migration, which may provide a target basis for treating human ovarian tumor with TFPI-2 protein therapy.

Objective To study the role of focal adhesion kinase in the breast carcinoma cell adhesion and migration mediated by coagulation factor Ⅶa combined with tissue factor to explore the mechanism of tissue factor promoting metastasis. Methods The count of adherent cells were detected by using colorimetric assay,cell migration was detected with modified Bodyen chamber,and the FAK and the tyrosine phosphorylation of FAK were detected by immunoprecipitation and Western blot.Results Factor Ⅶa combinaed with tissue factor induced the tyrosine phosphorylation of FAK,which took part in the cancer cell's migration.Conclusion FAK is an important message of the breast carcinoma's adhesion and migration mediated by TF.

Objective To study the apoptosis mechanism of K562 cell lines induced by mangiferin. Methods The mRNA expression levels of apoptosis-related genes including bcl-2, bax, survivin of K562 cells treated by mangiferin (25—200 ?mol/L) for 24, 48, 72, and 96 h were determined by RT-PCR; the BCR/ABL protein P210 level was detected by Western blotting. Results Mangiferin up-regulated bax gene of K562 cells significantly and down-regulated bcl-2 gene slightly, resulting in an enhancement of the ratio of bax/bcl-2. Mangiferin down-regulated the expression levels of P210 in K562 cells in a time-and concentration-dependent manner and so is the expression level of survivin mRNA in K562 cells. ConclusionThe mechanism of mangiferin-induced apoptosis in K562 leukemic cells might be involved in up-regulating the gene expression of bax and down-regulating the mRNA expression of BCR/ABL protein P210, bcl-2, and survivin.

Objective: To understand the effect of Carvedilol on mitochondrial oxidative phosphorylation function during the development of pressure overload induced left ventricular hypertrophy in rats and its mechanism.Methods: Male SD rats were randomized into 6 groups: 5-and 15-week coarctation of the abdominal aorta(H5,H15),5-and 15-week Carvedilol intervention(HR5,HR15) and 5-and 15-week sham operation(S5,S15).Hemodynamics and ventricular remodeling parameters were measured,and the mitochondrial respiratory function was detected by Clark oxygen electrode.Results: Compared with S5,mitochondrial state 3 and 4 respiration and the oxidative phosphorylation rate(OPR) were increased and the respiratory control rate(RCR) decreased significantly in the H5 group.In comparison with S15,state 3 respiration,OPR and RCR were reduced significantly in the H15group.Carvedilol increased the three parameters and restored them to the level of the S15.Conclusion: Mitochondrial respiratory function decreased during the development of pressure overload induced left ventricular hypertrophy in rats.Carvedilol could protect mitochondrial respiratory function and improve myocardial energy metabolism,which might be a mechanism underlying its protective effect on myocardium.

Objective To study the dosimetry parameters of 125I seed source (type Sinko BT-125-1) with thermoluminescent dosimeter (TLD) in the phantom.Methods The new type of phantom was modified to suit to measurement of a common type of 125I seed source.The AAPM TG43 protocol recommended measurements of dose-rate constant (Λ),radial dose function (gL (r)),and anisotropy function (F (r,θ)) have been performed in the phantom with TLD.Results The Λ was 0.928 cGyh-1 U-1.The gL(r) was determined at different radial distances r ranging from 1.0 to 10.0 cm with an interval of 1.0 cm ; and F (r,θ) at angles from 0° to 90° in 10° increments.The gL (r) of 125I seed source showed a difference of 9.6％ at the most in comparison to the corresponding values of 125I seed source (type Amersham 6711).The difference in F(2 cm,θ) of 125I seed source and Amersham 6711 was up to 10.2％ near the source end.With the phantom the combined standard uncertainty in the whole measurement was less than 6.0％.Conclusions The experimental results exhibit fairly small measurement uncertainties and good self-consistency.It's feasible to measure the dosimetry characters of permanent implant seeds in the modified phantom.

Objective To observe the changes of coagulation function in patients with acute lowerlimb deep venous thrombosis(DVT)and evaluate the risk factors for DVT. Methods Plasma APTT.PT,TT,D-dimer and fibrinogen(Fbg)were detected by an automated coagulation analyzer in 62 acute lower-limb DVT patients and 70 controls:Retrospective studies on the clinical data of all patients were done by binary logistic regression analysis.Results (1)In DVT group,plasma APTT,PT and TT,the levels of D-dimer and fibrinogen.and D-dimer/fibrinogen ratio(D/F ratio)were higher when compared with control group(au P＜0.01);(2)There were positive correlations between D-dimer and fibrinogen both in DVT and control groups(r=0.475,P＜0.01;r=0.564,P＜0.01,respectively);(3)Logistic analysis indicated that acute lower-limb DVT was associated with the presence of hypertension and increased plasma level of fibrinogen(OR=24.99,P＜0.01: OR=4.346.P＜0.01,respectively).Conclusions Hypertension and elevated plasma level of fibrinogen are independent risk factors for acute lower-limb DVT.

In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Annexin A2/*pharmacology ; Cells, Cultured ; Endothelium, Vascular/cytology ; Endothelium, Vascular/*metabolism ; Fibrinolysis ; Plasminogen/*metabolism ; Recombinant Proteins/pharmacology ; Tissue Plasminogen Activator/*metabolism ; Umbilical Veins/cytology

In order to study the role of annexin II, a recombinant expression vector, pZeoSV2(+) ANN II, containing the annexin II cDNA, was developed. The 1.1-kb-length annexin II cDNA was inserted into a expression vector, PZeoSV(+) and transfected into HL-60 cells which had low baseline expression of Ann- II. pZeoSV(+) ANN II was analyzed by restriction mapping and the Ann- II sequence identified. The ability of the transfected cells, non-transfected and mock-transfected cells to stimulate t-PA-depend plasminogen activation was compared. The results showed that HL-60 with pZeoSV(+) ANN II transfection could significantly increase the plasminogen activation (8.9 +/- 1.2 U) in vitro with the difference being significant as compared with non-transfected (1.5 +/- 0.4 U) and mock-transfected cells (4.2 +/- 0.9 U), respectively. Antiannexin II oligonucleotides significantly inhibited the binding ability of t-PA and plasminogen to annexin II, and obviously reduced the plasminogen activation in vitro. The above findings showed human umbilical vein endothelial cells (HUVECs) treated with sense or missense oligonucleotides indicated no significant change in binding of t-PA and PLG. Treatment of HUVECs with antiannexin II oligonucleotides could significantly reduce the plasminogen activation by 2.4 +/- 0.3 U as compared with sense oligonucleotide group in binding of t-PA and PLG. These results, therefore, suggest that Ann- II can bind plasminogen and participate in the stimulation of t-PA-dependent activation of plasminogen, and that interference with Ann-II mRNA by antisense oligonucleotide may be a new strategy for the therapy of bleeding in patients with hyperfibrinolysis.
Annexin A2 ; genetics ; metabolism ; physiology ; DNA, Complementary ; genetics ; Endothelium, Vascular ; cytology ; HL-60 Cells ; pathology ; Humans ; Oligonucleotides, Antisense ; genetics ; metabolism ; Plasminogen ; metabolism ; RNA, Messenger ; genetics ; metabolism ; physiology ; Receptors, Cell Surface ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Tissue Plasminogen Activator ; metabolism ; Transfection ; Umbilical Veins ; cytology

In order to further investigate the effect of annexin II (Ann-II) on tissue plasminogen activator (t-PA)-dependent plasminogen (PLG) activation and its interactive mechanism, recombinant native Ann-II bound t-PA, PLG and plasmin with high affinity was examined. The flow cytometric assay showed that the ann-II expression rate was higher in the human umbilical vein endothelial cell (HUVEC) (87.65%) than in the HL-60 cells as controls (35.79%). Two irrelevant proteins, bovine serum albumin (BSA) and equine IgG (EIG) had no effect on the production of plasmin. Ann-II-mediated enhancement of t-PA-dependent PLG activation was inhibited by epsilon-aminocaproic acid or by pretreatment of Ann-II with carboxypeptidase B with the inhibitive rate being 77.8% and 77.0%, respectively. It was revealed that the effect of Ann-II on PLG activation was specific for t-PA. Urokinase didn't bind to Ann-II, demonstrating the role of receptor-related lysine residues on activation of PLG, showing that the Ann-II-PLG interaction was dependent upon carboxyl-terminal lysine residues. These findings suggest that annexin II-mediated co-assembly of t-PA and PLG may promote plasmin generation and play a key role in modulating fibrinolysis on the endothelial surface.
Annexin A2 ; pharmacology ; Cells, Cultured ; Endothelium, Vascular ; cytology ; metabolism ; Fibrinolysis ; Humans ; Plasminogen ; metabolism ; Recombinant Proteins ; pharmacology ; Tissue Plasminogen Activator ; metabolism ; Umbilical Veins ; cytology

OBJECTIVETo explore a new method of alleviating graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT) through selected elimination of mouse alloreactive T cells (ARTC) by Fas-Fas ligand (FasL) passway.

METHODSThe Sca-1(+) early hematopoietic cells (EHCs) were isolated from BALB/c mouse (H-2(d)) bone marrow mononuclear cells (BMMC) by using a high gradient magnetic cell sorting system (MACS), then transferred with exogenous mouse FasL (mFasL) gene by retroviral gene transfecting technique. Afterward the transduced EHCs were expanded in vitro for one week followed by coculture with the spleen cells from BAC mouse (H-2(d) x b) as one-way mixed lymphocyte culture (OWMLC) for 6 days, then the cytotoxicity of treated BAC mouse spleen cells against Na(2)(51)CrO(4) labelling spleen cells from BALB/c mouse was observed.

RESULTSThe Sca-1(+) EHCs were successfully isolated by MACS, with a purity of (89.0 +/- 6.1)%. After transferred with exogenous mFasL gene and expanded for one week, the transferred EHCs in the 6 day OWMLC with the spleen cells from BAC mouse at a ratio of five to one resulted in an obvious inhibition of the BAC mouse spleen cells cytotoxicity against the BALB/c mouse spleen cell at different effector/target ratios as compared to the control group (P < 0.01).

CONCLUSIONThe higher exogenous mFasL-expressing mouse EHCs can deplete ARTC against their own major histocompatibility complex (MHC) antigens in vitro.

Animals ; Antigens, Ly ; immunology ; Fas Ligand Protein ; Female ; Graft vs Host Disease ; immunology ; Hematopoietic Stem Cell Transplantation ; Hematopoietic Stem Cells ; cytology ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Membrane Proteins ; immunology ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Signal Transduction ; Spleen ; cytology ; immunology ; T-Lymphocytes ; immunology ; Transfection