Adult human bone marrow-derived mesenchymal stem cells (MSCs) were cultured on microcarriers in spinner flasks and were compared with those in conventional culture in 12-well plates. For the production of adherently growing MSCs, macroporous CultiSpher G gelatin microcarriers were used in concentration of 1 g/L. The cells were seeded in a density of 5 x 10(4) cells/ml in both spinner culture and conventional stationary culture. The result showed that after 7 days of cultivation a maximum viable cell concentration of 5.15 x 10(5) cells/ml was obtained in spinner culture. Whereas the cell density increased to a maximum of 1.675 x 10(5) cells/ml on day 5 in conventional stationary culture. Lactate was produced up to 12.06 mmol/L in spinner culture and up to 13.10 mmol/L in stationary culture, and glucose was consumed up to 7.38 mmol/L and 5.37 mmol/L respectively. The average lactate yield on glucose consumption in spinner culture was only 1.63, lower than that in stationary culture 2.44. This indicated that the energy metabolism in spinner culture was significantly more efficient than that in conventional culture. After spinner culture for 12 days, the MSCs maintain the characteristics of stem cells. It is concluded that the microcarrier culture system is a suitable way to expand the seeding cells for tissue engineering.
Adult ; Antigens, CD ; analysis ; Bone Marrow Cells ; cytology ; metabolism ; ultrastructure ; Cell Count ; Cell Culture Techniques ; instrumentation ; methods ; Cell Division ; Cell Survival ; Flow Cytometry ; Glucose ; metabolism ; HLA-DR Antigens ; analysis ; Humans ; Lactates ; metabolism ; Mesoderm ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; Microscopy, Electron, Scanning ; Stem Cells ; cytology ; metabolism ; ultrastructure ; Time Factors

Adhesion to extracellular matrix plays important roles in the regulation of survival, proliferation, differentiation and homing of hematopoietic cells and is regulated by a wide variety of growth factors, adhesion receptors and other ligands that mediate the cell to matrix and cell to cell interaction. Stem cell factor (SCF) plays important roles in the regulating growth and self-renewal of hematopoietic stem/progenitor cells. In the report, the effects of stem cell factor on the adhesion of hematopoietic cells to fibronectin were observed by using a hematopoietic growth factor dependent cell line-Mo7e. Results showed that Mo7e cells express the very late antigen VLA-4 (beta1 alpha4) and VLA-5 (beta1 alpha5) integrins. The expression of the SCF receptor (c-kit) was also detected in the Mo7e cells. SCF enhances the adhesion of Mo7e cells to fibronectin in a concentration dependent manner. SCF enhanced adhesion of Mo7e cells to fibronectin was blocked by anti-beta1, alpha4 and alpha5 antibodies. Addition of PI-3 kinase inhibitors also blocked the adhesion of Mo7e cells to fibronectin induced by SCF. It was concluded that SCF enhances the adhesion of Mo7e cells to fibronectin, and this process is mediated by integrins and PI-3 kinase pathway.
Antibodies, Monoclonal ; pharmacology ; Cell Adhesion ; drug effects ; Chromones ; pharmacology ; Dose-Response Relationship, Drug ; Enzyme Inhibitors ; pharmacology ; Fibronectins ; metabolism ; Hematopoietic Stem Cells ; drug effects ; metabolism ; Humans ; Integrin alpha4beta1 ; immunology ; metabolism ; Integrin alpha5beta1 ; immunology ; metabolism ; Morpholines ; pharmacology ; Phosphatidylinositol 3-Kinases ; antagonists & inhibitors ; metabolism ; Stem Cell Factor ; pharmacology ; Tumor Cells, Cultured

Natural hirudin extracted from the secretion of medical leech salivary gland is a single-chain peptide containing 65 aminoacid residues with molecular weight of 7000 D, and exists in three isomers of HV1, HV2 and HV3. Hirudin possesses three disulfide bridges forming the structure of core cyclic peptides, which binds to the catalytic site of thrombin so as to inhibit the catalysis of thrombin. Its c-terminus rich in acidic aminoacid residues possesses hydrophilicity, and is free on the molecular surface, and can bind with fibrin recognition site of hirudin. The minimal segment of 12 - 16 C-terminal acidic residues keeps the minimal activity of anti-thrombosis. Thus, hirudin, as a potent and specific inhibitor of thrombin, can be used to protect from and to treat clinically thrombosis. As it has some disadvantages such as short half-life, bleeding side-effect and mono-function, and so on, hirudin has been fused with some other functional proteins in recent years. The obtained fusion proteins can prolong the half life of hirudin, or relieve it bleeding side effect, or bring new functions, such as thrombolysis, inhibiting the platelet aggregation, targeting specifically. The research progress in hirudin fusion protein was summarized in this review.
Anticoagulants ; pharmacology ; Delayed-Action Preparations ; Drug Delivery Systems ; Glucokinase ; biosynthesis ; genetics ; pharmacology ; Hirudins ; biosynthesis ; genetics ; pharmacology ; Humans ; Platelet Aggregation Inhibitors ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; pharmacology

Notch signal path plays important roles in the regulation of proliferation and differentiation of hematopoietic stem cells. An extracellular domain of human Delta-like-1 (hDll-1(ext)), one of Notch ligands, was cloned and expressed in CHO cells, and the effect of hDll-1(ext) on expansion of hematopoietic stem/progenitor cells was investigated in this study. Total RNA was isolated from human marrow mononuclear cells. hDll-1(ext) was amplified by RT-PCR and cloned to T vector, then the gene was sequenced and subcloned to pcDNA3.1/Myc-His(+)A expression vector. The constructed plasmid was transfected into CHO cells with lipofectin and the expression of secreted hDll-1(ext) in G418-resistant clones was assayed by Western blot. hDll-1(ext) high-expressed clone was cultured to collect supernatant. Fusion protein hDll-1(ext) was purified from the supernatant by immobilized metal affinity chromatography (IMAC). The results showed that expression of Notch-1 receptor was detected in cord blood-derived CD34(+) cells by RT-PCR. Human umbilical blood CD34(+) cells were cultured in serum-free medium containing SCF, IL-3, VEGF, and with or without purified hDll-1(ext) for 4 or 8 days. Effect of hDll-1(ext) on the expansion of progenitor cells was analyzed then by clonogenic assays. The number of CFU-Mix and HPP-CFC generated from the culture system containing hDll-1(ext) was 1.5 times of that from the control. In conclusion, the recombinant hDll-1(ext) promotes the expansion of primitive hematopoietic progenitors.
Animals ; Antigens, CD34 ; immunology ; Binding Sites ; genetics ; CHO Cells ; Cell Division ; drug effects ; physiology ; Colony-Forming Units Assay ; Cricetinae ; Endothelial Growth Factors ; pharmacology ; Fetal Blood ; cytology ; immunology ; metabolism ; Gene Expression ; Genetic Vectors ; genetics ; Glycoproteins ; genetics ; pharmacology ; physiology ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans ; Intercellular Signaling Peptides and Proteins ; pharmacology ; Interleukin-3 ; pharmacology ; Lymphokines ; pharmacology ; Membrane Proteins ; genetics ; RNA ; genetics ; metabolism ; Receptor, Notch1 ; Receptors, Cell Surface ; Recombinant Proteins ; isolation & purification ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cell Factor ; pharmacology ; Transcription Factors ; Transfection ; Vascular Endothelial Growth Factor A ; Vascular Endothelial Growth Factors

OBJECTIVETo evaluate the effects of hepatocyte growth factor (HGF) on the prevention of scar formation and the promotion of wound healing by gene transfer.

METHODSA total of 12 female New Zealand rabbits were used in this study. Rabbits were anesthetized with an intravenous injection of sodium pentobarbital, and identical wounds were made over the ventral surface of each ear. Five circular wounds, 7 mm in diameter, were created in each ear by excision through the skin to the underlying cartilage using sterile technique. After the surgical procedures, 10 of the rabbits were randomly allocated to five groups, with 2 rabbits in each group: Ad-HGF group 1, Ad-HGF group 2, Ad-HGF group 3, Ad-GFP (a reporter gene) group and the solvent group. Immediately after surgery, 6 x 10(7) pfu Ad-HGF, 6 x 10(8) pfu Ad-HGF, 6 x 10(9) pfu of Ad-HGF, 6 x 10(9) pfu of Ad-GFP, or same volume of solvent (PBS, pH 7.2) was applied once to each wound in groups 1 to 5, respectively. One additional rabbit was used to evaluate the transfer efficiency of the adenovirus vector by transferring Ad-GFP (6 x 10(9) pfu) into its wounds. Ice slides of wounds from this animal were observed under fluorescence microscopy. Another additional rabbit was used to evaluate the expression of HGF and TGFbeta1 after transferring Ad-HGF (6 x 10(9) pfu) into each of its wound. Immunohistochemistry was used for detection.

RESULTSThe effect of HGF on reducing excessive dermal scarring was observed by adenovirus-mediated gene transfer. Transfection of the human HGF cDNA into skin wounds through an adenoviral vector suppressed the over-expression of TGFbeta1, which plays an essential role in the progression of dermal fibrogenesis. Application of HGF to the wounds significantly enhanced wound healing and inhibited over scarring.

CONCLUSIONHGF gene therapy could be a new approach for preventing excessive dermal scarring in wound healing.

Animals ; Cicatrix ; prevention & control ; Female ; Gene Transfer Techniques ; Hepatocyte Growth Factor ; pharmacology ; Rabbits ; Random Allocation ; Wound Healing ; drug effects ; physiology

Membrane microparticles are shed from the plasma membrane of most eukaryotic cells when these cells were undergone activation or apoptosis, and released into the extracellular environment. Their composition depends on the cellular origin and processes triggering their formation. Several lines of evidence suggest that membrane microparticles might be able to facilitate cell-cell cross-talk and play an important roles in the regulation of survival, proliferation, differentiation, adhesion and chemotaxis of hematopoietic cells. Here, the components, mechanism of formation and the regulatory roles of membrane microparticles in hematopoiesis were reviewed.
Caveolae ; metabolism ; physiology ; Cell Membrane ; metabolism ; physiology ; Hematopoiesis ; physiology ; Humans ; Models, Biological ; R-SNARE Proteins ; metabolism ; physiology

Objective To investigate the therapeutic effect of interleukin-2(IL-2)on experimental autoimmune encephalomyelitis(EAE)mice.Methods After establishment of the EAE(experimental autoimmune encephalomyelitis) mouse models with MOG35-55 polypeptides,the mice were grouped according to the neurological function score and divided into control group,EAE group and low dose IL-2 treatment group.A double blind method was used to evaluate the neuro-logical impairment in mice.On the 29th day,pathological experiments were carried out in the mice's brain and spinal cord, hematoxylin-eosin staining was used to evaluate the scoring of inflammatory cell infiltration and luxol fast blue staining was used to evaluate the scoring of demyelinating.The proportion of regulatory T cells(Treg)and NK cells(natural killer cell, NK)was detected by flow cytometry,and the immunohistochemical method was used to detect the expressions of glial fibril -lary acidic protein(GFAP)and myelin basic protein(MBP)in the spinal cord.Results Compared with the EAE group, the neurological function score, the inflammatory cell infiltration score and the demyelinating score of the low dose IL-2 treatment group were reduced.The proportion of Treg cells in the low dose IL-2 treatment group was significantly higher than that in the EAE group,and the proportion of NK cells in the low dose IL-2 treatment group was slightly higher than that in the EAE group The expression of GFAP and MBP was detected by immunohistochemistry.The expression level of GFAP in low dose IL-2 treatment group was significantly lower than that in the EAE group,while the expression level of MBP was higher than that in the EAE group.Conclusion Low dose IL-2 has significant therapeutic effect on EAE mice.

OBJECTIVE To explore the antitumor effects of combined tanshinoneⅠ(TanⅠ),metformin (Met) and aspirin (Asp) on malignant melanoma in mice and the possible mechanisms. METHODS C57BL/6 mice were injected with 0.1 mL B16F10 cells(2.8×109L-1)to establish the subcutaneous trans-plantation tumor model at the right forelimbs axillary.Then,the mice were divided into 8 groups according to body mass,including model group, TanⅠgroup(20 mg·kg-1,ip),Asp group(210 mg·kg-1,orally in drinking water), Met group (70 mg·kg-1, orally in drinking water), Asp+Met group, TanⅠ+Asp group, TanⅠ+Met group and TanⅠ+Asp+Met group,10 mice in each group.Each mouse drank about 7 mL of water every day for a total of 18 d.The mouse body mass was measured every other day and the tumor diameter was calculated every day. The mice were sacrificed after treatment, the tumor mass was measured and the tumor inhibitory rates were counted. The histopathological changes of the liver and spleen were observed with HE staining. The percentage of lymphocytes in the tumor tissue such as CD8+T,CD4+T and Treg cells was detected by flow cytometry.Inflammatory factors such as interleukin-6 (IL-6),IL-1β and tumor necrosis factor-α (TNF-α) were detected by ELISA. RESULTS The body mass (including tumor mass)of mice in different groups increased during the experiment,but that of TanⅠ+Asp+Met group increased more slower than in model group(P<0.01).At the end of the experiment,no lesions were seen in any liver or spleen tissue by pathological observation,and the number of survivors was 8/10(model group),8/10(TanⅠgroup),7/10(Asp group),7/10(Met group),8/10(TanⅠ+Asp group), 8/10 (TanⅠ+Met group), 7/10 (Asp+Met group) and 5/10 (TanⅠ+Asp+ Met group), respectively. Compared with model group,there were no obvious changes in tumor volume or tumor mass in TanⅠ, Asp and Met groups and other two-two joint groups,but the tumor volume and tumor mass in TanⅠ+Asp+ Met group were significantly decreased (P<0.01, P<0.05), and the tumor inhibitory rate in this group was 46.2%.Compared with the model group,the percentage of CD8+T cells increased(P<0.05) in TanⅠ+Asp+Met group,but there were no significant changes in other groups.The contents of IL-6, IL-1β and TNF-α in tumor tissue of TanⅠ+Met group were much higher than in model group(P<0.01, P<0.05,P<0.05)and the content of IL-6 increased in TanⅠ+Asp+Met group(P<0.01).CONCLUSION Combination of TanⅠ,Asp and Met can effectively inhibit the growth of melanoma in mice,which may be related to the increasing percentage of CD8+T lymphocytes and IL-6 in tumor tissue.However there are possibly some side effects.

The proliferation and apoptosis of multiple myeloma (MM) cells were regulated by bone marrow microenvironments in which Notch signal plays important role in mediating cell-cell communication. However, the regulatory effect of Notch signal on the proliferation and apoptosis of multiple myeloma cells remains unclear. In this study, regulatory effect of Notch signal on the apoptosis of MM cells induced by DMS (N, N-dimethylsphingosine) was investigated. RT-PCR was used to identify the expression of Notch receptor and related molecules such as Dll-1, Jagged-1, Deltex-1 in MM cell lines. The intracellular domain of Notch (ICN), active form of Notch, was transferred into MM cells by retrovirus. The apoptosis of MM cells was determined by trypan blue exclusion tests and TdT-mediated dUTP nick end labeling (TUNEL) assay. The results showed that multiple myeloma cells expressed the Notch-1 and its related molecules. Notch activated multiple myeloma cell lines were obtained. Activation of Notch protected the multiple myeloma cells from the apoptosis induced by DMS,which was determined by cell viability and TUNEL assay. In conclusion, Notch signal suppressed the apoptosis of multiple myeloma cells and would possibly be a novel therapeutic target.
Apoptosis ; Cell Division ; Humans ; Multiple Myeloma ; drug therapy ; pathology ; Receptor, Notch1 ; Receptors, Cell Surface ; physiology ; Signal Transduction ; Transcription Factors ; physiology

AIMTo assess whether hepatocyte growth factor recruits bone marrow-derived endothelial progenitor cells into blood circulation to participate in postnatal angiogenesis and endothelium repair.

METHODSThe adenovirus vector encoding HGF gene (Ad-HGF) were intravenous administrated into BALB/c mice, and then serum HGF was determined by enzyme-linked immunosorbent assay, the number of CD34+ cells in peripheral blood was assayed by flow cytometry, and the nucleated cells in peripheral blood were isolated, cultured and the endothelial cell colonies were characterized by staining with antibodies against tie-2, vWF. The carbon tetrachloride-induced liver damage model of female mice was established. The peripheral blood nucleated cells of Ad-HGF treated male mice were intravenous administrated into these mice, and 4 weeks later, in situ hybridization for the sry gene was used to identify the implanted cells in the damaged tissues.

RESULTSIntravenous administration of Ad-HGF resulted in significant elevation of serum hepatocyte growth factor level and induced profoundly increase of endothelial progenitor cells in the peripheral blood, which were characterized by their ability to form endothelial cell colonies in culture and expression of CD34, tie-2, and vW factor. HGF-mobilized endothelial progenitors could incorporate into sites of neovascularization in a liver regeneration model.

CONCLUSIONHepatocyte growth factor could markedly recruit bone marrow-derived endothelial progenitor cells into blood circulation.

Animals ; Bone Marrow Cells ; cytology ; Endothelial Cells ; cytology ; Female ; Hematopoietic Stem Cell Mobilization ; Hepatocyte Growth Factor ; pharmacology ; Male ; Mice ; Mice, Inbred BALB C ; Stem Cells ; cytology