The purpose of this study was to devise an expanded ischemic flap model and to investigate the role of AMD-3100 (Plerixafor, chemokine receptor 4 inhibitor) in this model by confirming its effect on mobilization of stem cells from the bone marrow. Male Sprague-Dawley rats were used as an animal research model. The mobilization of stem cells from the bone marrow was confirmed in the AMD-3100-treated group. The fractions of endothelial progenitor cells (EPC) and the vascular endothelial growth factor receptor (VEGFR) 2+ cells in the peripheral blood were increased in groups treated with AMD-3100. The expression of vascular endothelial growth factor (VEGF) was increased in response to expansion or AMD injection. The expression of stromal cell derived factor (SDF)-1 and VEGFR2 were increased only in unexpanded flap treated with AMD-3100. Treatment with AMD-3100 increased both the number and area of blood vessels. However, there were no statistically significant differences in the survival area or physiologic microcirculation in rats from the other groups. This endogenous neovascularization induced by AMD-3100 may be a result of the increase in both the area and number of vessels, as well as paracrine augmentation of the expression of VEGF and EPCs. However, the presence of a tissue expander under the flap could block the neovascularization between the flap and the recipient regardless of AMD-3100 treatment and expansion.
Animals ; Anti-HIV Agents/pharmacology ; Bone Marrow Cells/cytology ; Chemokine CXCL12/biosynthesis ; Endothelial Progenitor Cells/*cytology ; Hematopoietic Stem Cells/*cytology ; Heterocyclic Compounds/*pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism ; Male ; Neovascularization, Physiologic ; Nitric Oxide Synthase Type III/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptors, CXCR4/antagonists & inhibitors ; Surgical Flaps/*blood supply/surgery ; Tissue Expansion/*methods ; Vascular Endothelial Growth Factor A/biosynthesis ; Vascular Endothelial Growth Factor Receptor-2/biosynthesis/metabolism

OBJECTIVETo construct a soluble prokaryotic expression vector of the CXCR7-specific antagonist SDF-1/54R and evaluate its activity.

METHODSSDF-1/54r gene amplified by PCR was inserted into the soluble expression vector pET-41a+ engineered with GST fusion tag, and the recombinant vector was transformed into E. coli strain BL21 (DE3). After IPTG induction of E. coli, the expressed recombinant protein was purified with GST affinity chromatography purification system and confirmed by SDS-PAGE and Western blotting assay. The target protein SDF-1/54R was obtained after digestion of the purified product with enterokinase. Breast cancer MCF-7 cells with high expression of CXCR7 was treated with SDF-1/54R and the cell proliferation and metastasis was evaluated with MTT and chemotaxis assays.

RESULTSThe target protein SDF-1/54R obtained showed an obvious inhibitory effect on the proliferation and metastasis of MCF-7 cells as confirmed by MTT and chemotaxis assays.

CONCLUSIONSDF-1/54R is a good antagonist of CXCR7 and shows a potential value as an effective anti-cancer agent.

Blotting, Western ; Chemokine CXCL12 ; metabolism ; Chromatography, Affinity ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Genetic Vectors ; Humans ; Polymerase Chain Reaction ; Receptors, CXCR ; antagonists & inhibitors ; Recombinant Proteins ; biosynthesis

OBJECTIVETo explore the effect of adenovirus-mediated human stromal cell-derived factor-1alpha (hSDF-1alpha) on ventricular remodeling in rats with myocardial infarction.

METHODSA recombinant adenoviral plasmid containing hSDF-1alpha cDNA was constructed using homologous recombination in bacteria and the recombinant adenovirus particles expressing hSDF-1alpha (AdV-SDF-1) were prepared. In rat models of myocardial infarction induced by left anterior descending artery occlusion, 1x10(10) PFU AdV-SDF-1 or PFU AdV-LacZ were injected at multiple sites into the infarcted myocardium 1 h after the operation, using 200 l cell-free PBS as the control. Four weeks after the injection, the cardiac function of the rats was analyzed, and the heart tissues were taken after the measurement of hemodynamics. On serial frozen sections, histological observation and morphometric measurement were carried out using a microscopic image analysis system, and the expression of hSDF-1alpha was detected by immunocytochemistry.

RESULTSFour weeks after AdV-SDF-1 injection, the myocardium in the infracted area showed significantly higher expression rates of hSDF-1alpha. The injection resulted in a obvious reduction in the infarct size and collagen content and a marked increase in the left ventricle wall, and the rats showed improved cardiac functions.

CONCLUSIONSDF-1alpha can improve the cardiac structure and function in rats with myocardial infarction by inhibiting collagen synthesis and deposition in the infarcted area.

Adenoviridae ; genetics ; metabolism ; Animals ; Chemokine CXCL12 ; administration & dosage ; biosynthesis ; genetics ; Female ; Gene Transfer Techniques ; Genetic Vectors ; genetics ; Male ; Myocardial Infarction ; physiopathology ; therapy ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; administration & dosage ; biosynthesis ; genetics ; Transfection ; Ventricular Remodeling ; drug effects

OBJECTIVETo construct the eukaryotic expression vector pDsRed2-N1-SDF-1alpha and observe its expression in the mouse bone marrow mesenchymal stem cells.

METHODSDF-1alpha gene sequence with XhoI, EcoRI restriction enzyme cutting site was amplified from the total RNA of mouse smooth muscle cells by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into the eukaryotic expression vector pDsRed2-N1 encoding red fluorescent protein gene, and the insertion was verified by endonuclease digestion and DNA sequencing. Mouse bone marrow mesenchymal stem cells identified with immunofluorescence assay for vimentin expression were transfected with the constructed plasmid pDsRed2-N1-SDF-1alpha, and the expression of sdf-1alpha was detected using immunofluorescence assay.

RESULTSThe DNA fragment amplified by PCR from the total RNA was identical to SDF-1alpha from the gene library, and an identical DNA fragment was also amplified from the recombinants. Sequence analysis confirmed the successful insertion of SDF-1alpha into the pDsRed2-N1 vector and the eukaryotic expression vector pDsRed2-N1-SDF-1alpha was successfully constructed. The cultured mouse bone marrow mesenchymal stem cells positive for vimentin protein showed SDF-1alpha expression 24 h after transfection with the recombinant vector.

CONCLUSIONThe pDsRed2-N1-SDF-1alpha eukaryotic expression vector constructed is capable of expression of SDF-1alpha fusion protein in the mouse bone marrow mesenchymal stem cells.

Animals ; Bone Marrow Cells ; cytology ; metabolism ; Chemokine CXCL12 ; biosynthesis ; genetics ; Female ; Genetic Vectors ; Mesenchymal Stromal Cells ; metabolism ; Mice ; Mice, Inbred C57BL ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Transfection

This study was amied to construct CXCR4 gene modified bone marrow mesenchymal stem cells (MSCs), and investigate the effect of CXCR4 expression on MSCs migration. The retrovirus vector pMSCV-CXCR4-IRES-GFP that expresses human CXCR4 gene was cloned,the MSCs were transduced by the virus, and the expression of OXCR4 was analyzed by FACS, RT-PCR and immunofluorescence staining. The migration assay was performed using Transwell method in the presence of SDF-1. FACS results showed that 46% of the transduced MSCs were CXCR4 positive, and 57% were GFP positive. The expression of CXCR4 in MSCs was also confirmed by RT-PCR and immunostaining. The migration of MSCs was induced by SDF-1 and was strongly dependent on CXCR4 expression. The concentration of SDF-1 had effect on the migration and the transmigration rate of CXCR4 modified; the amount of MSCs was 5-fold higher than that of untransduced MSCs when the optimal concentration rose to 50 ng/ml. These data indicate that SDF-1/CXCR4 plays an important role in MSCs migration ,and the CXCR4 genetic modification approach could be applied to enhance cell homing, and engraftment in MSCs therapy.
Bone Marrow Cells ; cytology ; Cell Movement ; Cells, Cultured ; Chemokine CXCL12 ; pharmacology ; Genetic Vectors ; genetics ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Receptors, CXCR4 ; biosynthesis ; genetics ; Retroviridae ; genetics ; metabolism ; Transduction, Genetic ; Transfection

This study was aimed to investigate the expression level of stromal cell derived factor-1 gene (SDF-1) in bone marrow mesenchymal stem cells (MSC) of patients with myelodysplastic syndrome (MDS). The MSC from bone marrow samples of MDS patients were isolated, cultured and expanded, the morphology and immunophenotype of MSC were analyzed. The expression levels of SDF-1 and internal reference GAPDH in MSC of MDS patients were detected by real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method and were compared with expression levels of healthy donors. The results showed that the expression levels of SDF-1 in MDS patients were significantly different from those in healthy donors (1.53 +/- 0.92 vs 5.51 +/- 0.99) (P < 0.01). SDF-1 gene expression levels in bone marrow MSC of MDS patients were significantly higher than that in MSC derived from healthy donors. It is concluded that the abnormal expression of SDF-1 gene in MSC may influence the regulation of hematopoiesis of the bone marrow microenvironment in MDS patients and it is worthy of further investigation for new clue on etiological mechanism and treatment of MDS.
Adult ; Aged ; Bone Marrow Cells ; metabolism ; pathology ; Cells, Cultured ; Chemokine CXCL12 ; biosynthesis ; genetics ; Female ; Humans ; Male ; Mesenchymal Stromal Cells ; metabolism ; pathology ; Middle Aged ; Myelodysplastic Syndromes ; genetics ; metabolism ; pathology

The study was aimed to explore the expression of stromal cell derived factor-1alpha (SDF-1alpha) and its receptor CXCR4, and their relationship with the extramedullary infiltration in acute lymphoblastic, grannulocytic and monocytic leukemia. 66 cases of acute leukemia included 31 cases of acute lymphoblatic leukemia (ALL), 20 cases of acute grannulocytic leukemia (M(2)) and 15 cases of acute monocytic leukemia (M(4)+M(5)). There were 41 cases with extramedullary infiltration and 25 cases without-extramedullary infiltration. Enzyme-linked immunoabsorbent assay (ELISA) and flow cytometry were used to determine expression of SDF-1alpha and CXCR4 respectively on leukemia cells in peripheral blood and bone marrow of different groups. The results showed that average plasma level of SDF-1alpha in the ALL, M(4)+M(5), M(2) patients and the normal control were 1317.87 +/- 220.76, 1339.79 +/- 187.06, 1063.70 +/- 190.74, 1908.34 +/- 135.55 (pg/ml) respectively. The average levels in the ALL, M(4)+M(5) and M(2) patients groups were lower than those in normal control group. Both levels in ALL and M(4)+M(5) patient groups were higher than that in M(2) patient group. The average levels of SDF-1alpha in patient group with extramedullary infiltration and patient groups without-extramedullary infiltration were 1252.49 +/- 263.12, 1234.91 +/- 185.50 (pg/ml) respectively. The former seemed as if higher than the latter, but without statistical significance. The MFI of CXCR4 expression in ALL, M(4)+M(5), M(2) patient group were 78.47 +/- 33.96, 67.21 +/- 24.29, 41.66 +/- 17.18, respectively. CXCR4 expression in ALL and M(4)+M(5) patient groups were higher than that in M(2) patient group (P > 0.05). There was no significant difference between the ALL and M(4)+M(5) patient group (P > 0.05). The MFI of CXCR4 expression in patients with extramedullary infiltration and patients without extramedullary infiltration were 81.72 +/- 27.63, 36.94 +/- 11.86 respectively. The former was higher than the latter (P < 0.05). It is concluded that the higher expression of CXCR4 on acute lymphoblatic and monocytic leukemia cells may be one of the molecular mechanisms of extramedullary infiltration in both kinds of leukemia. The average plasma levels of SDF-1alpha decreased in leukemia patients and this decrease not related to the extramedullar infiltration, which may be due to the SDF-1alpha local expression in the organ infiltrated.
Acute Disease ; Adolescent ; Adult ; Chemokine CXCL12 ; Chemokines, CXC ; biosynthesis ; genetics ; Child ; Child, Preschool ; Female ; Humans ; Leukemia ; metabolism ; pathology ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Leukemic Infiltration ; Male ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; metabolism ; pathology ; Receptors, CXCR4 ; biosynthesis ; genetics ; Stromal Cells ; metabolism

The stromal cell-derived factor 1 (SDF-1) interacts with its receptor CXCR4 to transduction signals, playing important roles in most physiological and pathological processes. It is reported that CXCR4 is highly expressed in many kinds of hematological malignancies and closely related to the prognosis, drug resistance and relapse of diseases. The growth of tumor cells can be inhibited by the anti-SDF-1 antibody or anti- CXCR4 antibody, supporting a new way for the therapy against hematological malignancies. Their expression in relation with prognosis and drug resistance of hematological malignancies are summarized in this review.
Chemokine CXCL12 ; biosynthesis ; genetics ; Drug Resistance, Neoplasm ; genetics ; Hematologic Neoplasms ; metabolism ; pathology ; Humans ; Prognosis ; Receptors, CXCR4 ; biosynthesis ; genetics ; Signal Transduction ; Stromal Cells ; metabolism

OBJECTIVETo study the expression of CXCR4 in acute leukemic cells and its clinical significance.

METHODBone marrow samples from 73 acute leukemia patients and leukemic cell lines were investigated by flow cytometry (FCM), the expression of SDF-1 in human marrow stromal cells and meninges were studied by using reverse transcription polymerase chain reaction (RT-PCR). Adhesion, migration and invasion of U937, NB4 and K562 cells were studied in vitro.

RESULTSThe expression rates of CXCR4 in ALL and AML patients was 65.6% and 17.1%, respectively. And it was 0.2%, 41.0% and 52.0% in K562, U937 and NB4 cells, respectively. The extramedullary infiltration rates were 61.9% and 18.2% for CXCR4 positive and negative groups of ALL, respectively (P < 0.05); while in AML, the number of peripheral white blood cells in CXCR4 positive group was lower than that in CXCR4 negative group (P < 0.05). SDF-1alpha could enhance the adhesion, migration and invasion capacity of leukemic cells in vitro.

CONCLUSIONOverexpression of CXCR4 in AL cells might be the molecular mechanism of extramedullary infiltration in leukemia.

Acute Disease ; Adolescent ; Adult ; Aged ; Bone Marrow Cells ; metabolism ; pathology ; Cell Adhesion ; Cell Line, Tumor ; Cell Movement ; Chemokine CXCL12 ; genetics ; Female ; Fibroblasts ; metabolism ; pathology ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; Humans ; K562 Cells ; Leukemia ; genetics ; metabolism ; pathology ; Leukemic Infiltration ; metabolism ; pathology ; Male ; Meninges ; metabolism ; pathology ; Middle Aged ; Receptors, CXCR4 ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; U937 Cells

OBJECTIVETo investigate the anti-HIV effects of ampelopsin and its interaction with HIV-1 coreceptor CXCR4.

METHODSThrough anti-virus experiments in vitro, the inhibitory effect of ampelopsin on HIV-1 infection was verified. Chemotaxis assay was performed to show the ability to induce PBMCs migration by ampelopsin, RANTES and SDF-1alpha. Fluorescence labelling monoclonal antibody was utilized to observe the interaction of ampelopsin and CXCR4. Mice immunosuppressant model was also established to detail the role ampelopsin played in regulating cellular immunological functions.

RESULTSAmpelopsin could protect sensitive cells against HIV-1 infection and dramatically reduce HIV-1 antigen P24 expression. HIV-1SF33 attaching to MT-4 cells was interfered by ampelopsin, and the EC50 was 0.175 mg/mL for cellular protection and 0.024 mg/mL for P24 inhibition. At co-cultivating phase, EC50 was 0.229 mg/mL and 0.197 mg/mL respectively. Furthermore, the EC50 was 0.179 mg/mL and 0.348 mg/mL in acute infection. Human PBMCs migration was induced after being challenged with ampelopsin or chemokines, and synergistic action was observed during co-treatment. Ampelopsin alone resulted in maximal chemotaxis at 1 mg/mL. HIV-1 co-receptor CXCR4 on the surface of PBMCs was decreased by internalization, which indicated the effect of ampelopsin on CXCR4. About 70% CXCR4 was reduced by ampelopsin at 1 mg/mL. Ampelopsin also augmented cellular immunological functions in immunosuppressive mice.

CONCLUSIONAmpelopsin displays a strong inhibitive role during HIV-1 absorption, incubation and acute infection. These results are coincident with its immune enhancement.

Ampelopsis ; chemistry ; Animals ; Anti-HIV Agents ; pharmacology ; Cell Line ; Chemokine CCL5 ; pharmacology ; Chemokine CXCL12 ; Chemokines, CXC ; pharmacology ; Chemotaxis, Leukocyte ; Down-Regulation ; Drugs, Chinese Herbal ; Flavonoids ; economics ; isolation & purification ; pharmacology ; HIV Infections ; virology ; HIV-1 ; drug effects ; metabolism ; pathogenicity ; Humans ; Interleukin-2 ; biosynthesis ; Leukocytes, Mononuclear ; drug effects ; Mice ; Mice, Inbred BALB C ; Models, Animal ; Plant Roots ; chemistry ; Receptors, CXCR4 ; antagonists & inhibitors ; drug effects ; Spleen ; immunology ; T-Lymphocytes ; immunology