To demonstrate the effect of AB serum on terminal erythroid differentiation ex vivo.
 Methods: After separation of CD34+ cells from cord blood, the cells were cultured and divided into a control group and an experimental group. The effects of AB serum were examined by the expressions of different markers (GPA, Band3 and α4-integrin) for erythroblast differentiation and enucleation by flow cytometry. 
 Results: The CD34+ cells were successfully differentiated to enucleated red blood cells. There were evident differences among the expressions of GPA, Band3 and α4-integrin between the 2 groups. The percentage of GPA positive cells in the experimental group was bigger than that in the control group in every time point. The expression of Band3 in the experimental group was higher than that in the control group. The expression of α4-integrin in the experimental group was lower than that in the control group. In addition, the enucleation rate in the experimental group was higher than that in the control group.
 Conclusion: AB serum can promote the cell differentiation and enucleation during terminal erythroid differentiation in vitro.
ABO Blood-Group System ; blood ; physiology ; Anion Exchange Protein 1, Erythrocyte ; metabolism ; Antigens, CD34 ; blood ; Cell Differentiation ; genetics ; physiology ; Cell Nucleus ; Cells, Cultured ; Erythrocytes ; physiology ; ultrastructure ; Erythropoiesis ; genetics ; physiology ; Fetal Blood ; cytology ; physiology ; Flow Cytometry ; Glycophorins ; metabolism ; Humans ; Integrin alpha4beta1 ; metabolism

OBJECTIVETo explore the influence of Acorus tatarinowii schott on ultrastructure and permeability of BBB.

METHODThe ultrastructure and permeability of BBB on rats with A. tatarinowii by electron microscope were observed. The even's blue (EB) and sodium phenytoin in brain was determined by UV and HPLC-MS.

RESULTAfter give A. tatarinowii, tight junctions of the endothelial cell opened in cotex, and the concentration of EB and sodium phenytoin in brain are significant increased.

CONCLUSIONA. tatarinowii can increase the permeability of BBB, and show its 'Kaiqiao' effect.

Acorus ; chemistry ; Animals ; Blood-Brain Barrier ; drug effects ; Brain ; cytology ; Cells, Cultured ; Cerebrovascular Circulation ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; Male ; Microscopy, Electron, Transmission ; Neurons ; diagnostic imaging ; drug effects ; Permeability ; drug effects ; Rats ; Rats, Sprague-Dawley ; Tight Junctions ; drug effects ; ultrastructure ; Ultrasonography

BACKGROUNDTumstatin is a recently developed endogenous vascular endothelial growth inhibitor that can be applied as an anti-angiogenesis and antineoplastic agent. The study aimed to design and synthesize the small molecular angiogenesis inhibition-related peptide (peptide 21), to replicate the structural and functional features of the active zone of angiogenesis inhibition using tumstatin and to prove that synthesized peptide 21 has a similar activity: specifically inhibiting tumor angiogenesis like tumstatin.

METHODSPeptide 21 was designed and synthesized using biological engineering technology. To determine its biological action, the human umbilical vein endothelial cell line ECV304, the human ovarian cancer cell line SKOV-3 and the mouse embryo-derived NIH3T3 fibroblasts were used in in vitro experiments to determine the effect of peptide 21 on proliferation of the three cell lines using the MTT test and growth curves. Transmission electron microscopy (TEM) and flow cytometry (FCM) were applied to analyze the peptide 21-induced apoptosis of the three cell lines qualitatively and quantitatively. In animal experiments, tumor models in nude mice subcutaneously grafted with SKOV-3 were used to observe the effects of peptide 21 on tumor weight, size and microvessel density (MVD). To initially investigate the role of peptide 21, the effect of peptide 21 on the expression of vascular endothelial growth factors (VEGFs) by tumor tissue was semi-quantitatively analyzed.

RESULTSThe in vitro MTT test and growth curves all indicated that cloned peptide 21 could specifically inhibit ECV304 proliferation in a dose-dependent manner (P < 0.01); TEM and FCM showed that peptide 21 could specifically induce ECV304 apoptosis (P < 0.01). Results of in vivo experiments showed that tumors in the peptide 21 group grew more slowly. The weight and size of the tumors after 21 days of treatment were smaller than those in the control group (P < 0.05), with a mean tumor inhibition rate of 67.86%; MVD of the tumor tissue in the peptide 21 group was significantly lower than in the control group (P < 0.05); the number of cells positive for VEGF in the peptide 21 group was significantly fewer than in the control group (P < 0.01).

CONCLUSIONSSimilar to the activity of tumstatin in specifically inhibiting tumor angiogenesis, peptide 21 may specifically inhibit tumor endothelial cell proliferation and induce their apoptosis, thereby suppressing tumor angiogenesis and indirectly inhibit the growth, infiltration and metastasis of tumors. Peptide 21 may exert its effect through down-regulating the VEGF expression of tumor cells and vascular endothelial cells.

Angiogenesis Inhibitors ; pharmacology ; Animals ; Apoptosis ; drug effects ; Autoantigens ; chemistry ; genetics ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Collagen Type IV ; chemistry ; genetics ; Dose-Response Relationship, Drug ; Endothelial Cells ; cytology ; drug effects ; ultrastructure ; Flow Cytometry ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Microscopy, Electron, Transmission ; NIH 3T3 Cells ; Neoplasms, Experimental ; blood supply ; pathology ; prevention & control ; Neovascularization, Pathologic ; pathology ; prevention & control ; Peptides ; chemistry ; genetics ; pharmacology ; Recombinant Proteins ; chemistry ; pharmacology ; Xenograft Model Antitumor Assays

OBJECTIVETo observe the histopathological changes of a novel small-caliber vascular graft after implantation in canine theca interna under scanning electron microscope.

METHODA 3 cm segment of the vascular graft (diameter of 4 mm) was implanted in an end-to-end fashion to bridge the severed carotid artery in 19 healthy dogs. Color Doppler sonography was performed 2 weeks after the operation to observe the patency rate of artificial blood vessel. At 1, 8, 12 and 24 week postimplantation, the arteries (4, 4, 6 and 5, respectively) were collected for optical and scanning electron microscopies after angiography to observe the patency of the arteries.

RESULTSOf the total of 19 arteries, occlusion occurred in 1 at 12 weeks and 1 at 24 weeks. Optical and electron microscopies showed that 1 week after implantation, slight fibroplasias and formation of red thrombus could be seen at the vascular anastomosis without endothelial cell lining. At 8 weeks, the host tissue grew into the lumen of the graft through the pores to form uniform neointima consisting of plenty of collagen fibers, but still without endothelial cells. At 12 weeks, discontinuous endothelial cells were seen to grow on the surface of the neointima. In the middle segment of the vascular graft, immature endothelial cells were found to grow in clusters. The structure of the neointima was loose in comparison with that at the anastomosis, with occasional inflammation cells. Twenty-four weeks after grafting, endothelial cells grew over the entire inner wall of the patent graft, and the surface of the neointima at the anastomosis was lined with continuous endothelial cells.

CONCLUSIONThe vascular graft can be useful for reconstruction of canine carotid artery defect and achieves good endothelialization 24 weeks after implantation.

Animals ; Blood Vessel Prosthesis ; Blood Vessel Prosthesis Implantation ; methods ; Carotid Arteries ; diagnostic imaging ; physiology ; surgery ; Collagen ; metabolism ; Dogs ; Endothelial Cells ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron, Scanning ; Models, Animal ; Time Factors ; Tunica Intima ; cytology ; metabolism ; ultrastructure ; Ultrasonography, Doppler, Color ; Vascular Patency

OBJECTIVETo explore the feasibility of the tissue engineered venous grafts (TEVGs) constructed in vitro based on canine autologous bone marrow-derived endothelial progenitor cells (EPCs) and porcine decellularized aortic scaffolds implanted into the canine inferior vena cava.

METHODSTo draw out a volume of 8 - 12 ml of bone marrow from the canine (n = 8), to culture and expand EPCs in vitro using conditioned medium. After labeled with a red fluorescent dye PKH26-GL, the cells were seeded onto the luminal surface of decellularized porcine scaffolds with single, rotative method for 4 h. Following static culture for 24 - 72 h, the hybrids were implanted to replace autologous canine inferior vena cava about 4 cm long. Meantime one femoral artery-venous shunt about 1 cm long was performed. The non-seeded decellularized scaffolds (n = 4) were performed the same as control. Angiography was performed and the hybrids were explanted for morphology and labeled cells' immuno-fluorescence examinations at postoperative 10 d, 4 weeks and 12 weeks, respectively.

RESULTSThe patent number of experiment (control) group were 7/7 (2/4), 6/6 (2/2) and 4/4 (1/2) at postoperative 10 d, 4 weeks and 12 weeks, respectively. At 12 weeks, tightly confluence endothelial cells which covered the whole inner luminal surface of the explants were detected by immunohistochemistry of factor VIII and scanning electron microscopy, while fibrin-based pseudo-intima was detected on the inner luminal surface of matrix in the solo patent dog from the control group. Meanwhile, fibroblasts and alpha-actin positive cells in the matrices were found by transmission electron microscopy and alpha-actin immunohistochemistry. PKH26-GL labeled EPCs sustained on the luminal surface at a rather proportion accompanied by newly formed endothelial cells. However, the explants in both groups showed partial stenosis.

CONCLUSIONSSuch constructed tissue engineered venous graft based on canine autologous bone marrow-derived endothelial progenitor cells and porcine decellularized aortic matrices is promising and deserve to further improvement and testing.

Actins ; analysis ; Animals ; Blood Vessel Prosthesis ; Blood Vessel Prosthesis Implantation ; Bone Marrow Cells ; cytology ; metabolism ; ultrastructure ; Cells, Cultured ; Dogs ; Endothelial Cells ; cytology ; metabolism ; ultrastructure ; Factor VIII ; analysis ; Feasibility Studies ; Hematopoietic Stem Cell Transplantation ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Stem Cells ; cytology ; metabolism ; ultrastructure ; Swine ; Tissue Engineering ; methods ; Transplantation, Autologous ; Veins ; Vena Cava, Inferior ; surgery

OBJECTIVETo study the influence of conditioned medium of rat brain microvascular endothelial cells on mitochondrial function of cortical neurons and the protective effect of Tongluo Jiunao Injection (TJI) on it.

METHODSFour kinds of conditioned endothelial cell (EC) cultured medium were prepared, i.e. the N-CM medium prepared by EC cultured in the normal conditioned medium without any treatment; the NT-CM prepared by EC cultured in N-CM and treated with TJI 1 microl/ml for 10 h; the I-CM prepared by EC cultured in the non-glucose kreb medium under hypoxia condition; and the IT-CM by EC pre-treatce with TJI 1 microl/ml for 4 h and cultured as that of I-CM. The levels of neuronic mitochondrial activity, membrane potential (MMP) and cytochrome C (Cyt C) were determined before and after the glucose-oxygen deprived model neurons of brain cortex being cultured with different kinds of conditioned EC cultured medium for assessing the effects of these media on mitochondria of injured neuron.

RESULTSAs compared with those of the normal neuron, the mitochondrial activity and MMP of all injured neurons decreased and Cyt C level increased significantly. But comparison of these indexes among neurons cultured with different conditioned EC culture media showed that the greatest extent abnormality revealed in the N-CM cultured neurons, which even greater than that in the model neuron; while that was less in the N-CM cutured neuron than in model neuron; as for those cultured in the NT-CM and IT-CM, i.e. the TJI treated cuture medium, the abnormal changes were reduced significantly when compared with those cultured in medium untreated with TJI (N-CM and I-CM), respectively (all P < 0.05).

CONCLUSIONThe paracrine secretion of the brain microvascular endothelial cells has evident regulatory effect on survival of the injured neurons, which might possibly be related to its protective effect on neuron mitochondrial function, and TJI could enhance the protective effect.

Animals ; Capillaries ; cytology ; Cells, Cultured ; Cerebral Cortex ; blood supply ; cytology ; Culture Media, Conditioned ; pharmacology ; Cytochromes c ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Endothelial Cells ; cytology ; drug effects ; ultrastructure ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; physiology ; Neurons ; cytology ; drug effects ; ultrastructure ; Rats

Hematological, cytochemical and ultrastructural features of blood cells in fishing cat (Felis viverrina) were evaluated using complete blood cell counts with routine and cytochemical blood stains, and scanning and transmission electron microscopy. No statistically significant difference was found in different genders of this animal. Unique features of blood cells in this animal were identified in hematological, cytochemical and ultrastructural studies. This study contributes to broaden hematological resources in wildlife animals and provides a guideline for identification of blood cells in the fishing cat.
Animals ; Animals, Zoo ; Blood Cells/*cytology/ultrastructure ; Felis/*blood ; Female ; Histocytochemistry/veterinary ; Male ; Microscopy, Electron, Scanning/veterinary ; Microscopy, Electron, Transmission/veterinary ; Thailand

AIMA method of coculture of brain capillary endothelial cells (BCECs) and astrocytes of rats was used to evaluate nanoparticle's blood-brain barrier (BBB) transcytosis and toxicity at the endothelial tight junction.

METHODSA lipophilic fluorescent probe, 6-coumarin, was incorporated in poly (ethyleneglycol)-poly (lactide) nanoparticle using double emulsion/solvent evaporation method. BCECs and astrocytes were firstly isolated from brain of newborn rats and characterized by their morphology and immunocytochemistry staining, separately. Subsequently, a coculture model with BCECs on the top of micro-porous membrane of cell culture insert and astrocytes on the bottom side was established. The permeability of 14C-labeled sucrose and nanoparticle were determined, separately.

RESULTSThe mean weight-based diameter of 6-coumarin loaded nanoparticles was (102.4 +/- 6.8) nm, with zeta potential of (-16.81 +/- 1.05) mV. BCECs were positive for factor VIII staining and glial fibrillary acidic protein was expressed in astrocytes. The transendothelial electrical resistance reached up to (313 +/- 23) omega x cm2. The tight junction between BCECs in the coculture model could be visualized by both scanning electron microscopy and transmission electron microscopy. The unchanged paracellular transport of sucrose proved that nanoparticle with concentration lower than 200 microg x mL(-1) did not impact the integrity of BBB endothelial tight junctions. The permeability of 10 microg x mL(-1) 6-coumarin labeled nanoparticle was 0.29 x 10(-3) cm x min(-1).

CONCLUSIONThis in vitro experimental model of rat BBB was close to resemble the in vivo situation for examination of the permeability of nanoparticle and toxicity evaluation.

Animals ; Animals, Newborn ; Astrocytes ; metabolism ; ultrastructure ; Biological Transport ; Blood-Brain Barrier ; Brain ; blood supply ; cytology ; Capillaries ; cytology ; Cell Membrane Permeability ; Coculture Techniques ; Coumarins ; administration & dosage ; pharmacokinetics ; toxicity ; Endothelial Cells ; metabolism ; ultrastructure ; Factor VIII ; metabolism ; Glial Fibrillary Acidic Protein ; metabolism ; Nanoparticles ; Polyesters ; Polyethylene Glycols ; Rats ; Rats, Sprague-Dawley ; Sucrose ; pharmacokinetics

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; genetics ; Cytarabine ; pharmacology ; Erythrocytes ; cytology ; metabolism ; ultrastructure ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Macrophages ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Time Factors

OBJECTIVETo construct the recombinant fowlpox virus (rFPV) coexpressing HIV-1 gag-gp120 and hIL-6.

METHODSThe recombinant expressing plasmid pUTA-GE-IL6 was successfully constructed by inserting gag-gp120 gene and hIL-6 gene into the downstream of the combined promoter ATI-p7.5 and p7.5 tandem promoter respectively. After transfecting the plasmid into chicken embryonic fibroblast (CEF) cells preinfected with FPV 282E4 strain and selecting the recombinant virus under the pressure of BUdR. The recombinant virus was analyzed by nucleic acid probe hybridization and immunoblotting. In addition, the formation of virus-like particle and the expression of interested proteins in the recombinant virus-infected p815 cells were observed, and the immunogenicity of the recombinant virus was also analyzed.

RESULTSThere was colorable dot for the positive recombinant virus, immunoblotting analysis showed that the recombinant virus could expressed both gag-gp120 and IL-6. Virus-like particles (VLP) were formed in virus-infected cells, and the interested proteins could be expressed in mammalian cells infected by the recombinant virus. The immunity index from the immunized mice showed that the recombinant virus had good immunogenicity.

CONCLUSIONThe recombinant fowlpox virus coexpressing gag-gp120 and IL-6 was successfully constructed, which may provide basis for the preparation of live vector genetic engineering vaccine and macromolecule particle vaccine against HIV-1.

Animals ; Antibodies, Viral ; blood ; Blotting, Western ; Cells, Cultured ; Chick Embryo ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Fibroblasts ; cytology ; metabolism ; ultrastructure ; Fowlpox ; blood ; immunology ; virology ; Fowlpox virus ; genetics ; immunology ; Gene Products, gag ; genetics ; metabolism ; Genetic Vectors ; genetics ; HIV Envelope Protein gp120 ; genetics ; metabolism ; HIV-1 ; genetics ; metabolism ; Immunization ; methods ; Interleukin-6 ; genetics ; metabolism ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Transfection ; Viral Vaccines ; genetics ; immunology ; metabolism