Objective To investigate the effects of hyaluronan(HA) on phagocytosis of macrophages and to explore the mechanism.Methods CD44 of macrophages was labeled with immunochemical method.In phagokinetic track assay,the areas without gold particles around the cells were measured with an image analyzer,shape of the cells was viewed under a scanning electron microscope.In fluorescent bead phagocytosis assay,the phagocytosed beads were conunted,the distribution of intracellular F-actin was viewed.In HA-coated bead phagocytosis assay,the phagocytosed beads were counted,shape of the cells was viewed with scanning electron microscope,the level of the intracellular Ca~(2+) was analyzed with confocal laser scanning microscope when the beads were phagocytosed.After treatment with anti-CD44 antibody,the changes of phagocytosis of the cells were examined.Results CD44 expressed on macrophages.The phagocytosing cells were round,their lamellipodia and filopodia were short.The membrane of the cells formed cup-like invagination when they engulfed particles and beads.In HA group,the phagocytosed gold particles and fluorescent beads increased.There was rich F-actin in the area where the fluorescent bead was engulfed.In HA-coated group,number of the phagocytosed HA-coated beads was great,the level of the intracellular Ca~(2+) in the area where the bead was engulfed increased significantly.After treatment with anti-CD44 antibody,the phagocytosed gold particles,fluorescent beads and HA-coated beads decreased.Conclusion HA promotes phagocytosis of macrophages,CD44 mediates effects of HA on phagocytosis of the cells.

Objective To study the changes of biological characteristics of CD34~+/CD133~+/VEGFR-3~+ lymphatic endothelial progenitor cells(EPCs) isolated from umbilical cord blood after induction with VEGF-C and investigate the mechanisms of EPCs differentiation toward lymphatic endothelial cells. Methods Mononuclear cells were isolated from umbilical cord blood using density centrifugation with Percoll solution.CD34~+/CD133~+/VEGFR-3~+ cells were sorted with FACS.Differentiation of the cells was induced with VEGF-C.The ultrastructural changes of the cells were viewed under scanning and transmission electron microscopes.Changes in expression of markers of the cells were viewed under confocal laser scanning microscope. Results Lymphatic EPCs isolated from umbilical cord blood expressed CD34,CD133 and VEGFR-3.At 7 day after induction with VEGF-C,CD34~+/CD133~+/VEGFR-3~+ cells were shuttle-like,there were a lamellipodium,numerous filopodia and many short microvilli.Caveolae were observed.Mitochondrion and rough endoplasmic reticulum were rich in cytoplasm.At 14 day after induction,the cells demonstrated appearance of the endothelial cell and expressed lymphatic endothelial specific makers LYVE-1 and 5'-nucleotidase,the expression of CD133 disappeared.Weibel-Palade body was observed in the cytoplasm of the cell.Conclusion There are CD34~+/CD133~+/VEGFR-3~+ lymphatic EPCs in human umbilical cord blood.The cells may differentiate into lymphatic endothelial cells through VEGF-C/VEGFR-3 signaling pathway after induction with VEGF-C.

Objective To investigate morphologic, structural and functional characteristics of cardiomyocytes from neonate rats and to set up a desirable technique for isolating and purifying the cardiomyocytes from neonate rats. Methods Using trypsin-digestion, mechanical separation, twice seedings and bromodeoxyuridine (BrdU)-treatment, the cardiomyocytes were isolated and purified from the hearts of neonate rats at 1-7 days after birth. Shapes and spontaneous pulsation of the cells were viewed. The cells were identified with cardiac isoform of tropnin T(cTnT) immunostaining. Ultrastructural features of the cells were examined with in situ transmission electron microscopy. Responses of the cells to adenine and isoprenaline were also examined. Results More than 95% cells isolated from the hearts of neonate rats are cardiomyocytes. The vital cells are more than 95%. Neonate rat cardiomyocytes include short columnar or rhabdoid cells and irregular cells. The most rhabdoid cells from the rats at 1-3 days after birth present the ultrastructural features of immature cardiomyocytes. The rhabdoid cells from the rats at 6-7 days after birth have some ultrastructural features of mature cardiomyocytes. Comparing with the cells at 1-3 days after birth, cTnT expression in the cells is slightly enhanced, the transverse striation was obvious. The irregular cells contain less bundles of myofilaments, the filaments are arranged irregularly. There are a few small cells which are in undifferentiated state. More than 80% cells show spontaneous pulsation at 72 hours after incubation. After treatment with adrenine and isoprenaline, the number of the cells with spontaneous pulsation increases and the intension of spontaneous pulsation is enhanced. The responses of the rhabdoid cells from the rats at 6-7 days after birth to adenine and isoprenaline are much stronger. Conclusion There are two kinds of neonate rat cardiomyocytes. They are different in ultrastructures, spontaneous pulsation and responses to adenine and isoprenaline. The cardiomyocytes from rats at 6-7 days after birth are suitable for experiments in vitro as mature cardiomyocyte. The method set up in this experiment is desirable for culture of neonate rat cardiomyocytes.

Objective To investigate the distrbution of actin and the level of Ca 2+ in macrophages activated with LPS and IFN\|? and study effects of reorganization of actin and the level of Ca 2+ on migration and phagocytosis of macrophages. Methods Rat macrophages were activated with LPS and INF\|?,actin and intracellular free Ca 2+ were labeled with phallacidin and Fura\|2,the distribution of actin and the level of Ca 2+ were observed and analyzed with Ca 2+ image analysis system and confocal laser scanning microscope. Results The migrating cell and the phagocytizing cells increased in the activated macrophages.F\|actin and the level of Ca 2+ of the activated macrophages increased.There was a front and a back in the migrating cell.Lamellipodia and filopodia protruded from the front.F\|actin of the lamellipodium and filopodium was rich.There were stress fibers of F\|actin at the bottom of the migrating cells.In the migrating macrophages,the level fo Ca 2+ in the back was high than that in the front.When the migrating cell turned their growth direction,the level of Ca 2+ at the curve increased greatly,especially in its lateral region.When macrophage phagocytized the dead lymphocyte,Ca 2+ in the phagocytizing region increased. Conclusion When macrophages are activated with LPS and IFN\|?,migration and phagocytosis increase greatly.Particular morphologic changes in the distribution of F\|actin and the level of intracellular free Ca 2+ occur. [

Objective In order to investigate effects of extracellular matrix on lymphangiogenesis. Methods Endothelial cells of canine thoracic duct were incubated in type I collagen gel and on Matrigel basement membrane matrix gel.Morphologic changes of endothelial cells and tube formation were observed under phase contrast microscope,structures of lymphaticlike channels were observed with transmission electron microscope,tube formation was quantified by measuring the length and area of tubes with image analyzer. Results Lymphatic endothelial cells on type Ⅰ collagen gel grew to confluent monolayers,no tubes were found.The cells underwent morphologic changes and reorganized into lymphaticlike tubes when the cells were covered with the top gel layer.Tubes of heparin\|treated dishes were more than that of the control dishes,tubes of dishes treated with heparin and bFGF together were more than that of dishes treated with heparin or bFGF alone.Endothelial cells on Matrigel basement membrane matrix gel migrated into the upper part of the gel and formed tubes.The tubes were thin,the cells became long.The tubes were in traction obviously.In ultrathin sections,tubes formed by lymphatic endothelial cells represented morphologic characteristics of lymphatic capillary.The lymphaticlike tubes contained matrix.Conclusion\ Endothelial cells of canine thoracic duct reorganized into lymphaticlike channels in type Ⅰ collagen gel and Matrigel basement membrane matrix gel.Extracellular matrix plays important roles in morphological changes and migration of lymphatic endothelial clells and lymphangiogenesis.\;[

Objective To compare features in expression of adhesion molecules of immunoglobin super family on lymphatic,vascular and microvascular endothelial cells and explore implications in expression of the adhesion molecules of immunoglobin super family on lymphatic endothelial cells. Methods Endothelial cells were isolated from canine thoracic ducts,common carotid arteries,internal jugular veins and pulmonary microvessels.Expression of PECAM-1,ICAM-1,ICAM-3,VCAM-1 and CD44 on all kinds of endothelial cells were examined by immunofluorescence staining and viewed using a fluorescence microscope and a confocal laser scanning microscope.Expression strength of the adhesion molecules was analyzed with an image analyzer. Results The arterial,venous and microvascular endothelial cells expressed PECAM-1,ICAM-1,ICAM-3,VCAM-1 and CD44.Expression of ICAM-1 and ICAM-3 on the cells was weak.Expression of VCAM-1 on the arterial and microvascular endothelial cells was stronger than that on venous endothelial cells.The lymphatic endothelial cells expressed PECAM-1,ICAM-1,ICAM-3 and CD44,expression of VCAM-1 was not observed.Expression of ICAM-3 and CD44 on the cells was stronger than that on vascular and microvascular endothelial cells.Conclusion\ In comparison with the arterial,venous,microvascular endothelial cells,lymphatic endothelial cells do not express VCAM-1,expression of ICAM-3 and CD44 is stronger.This is helpful to explain mechanisms of adhesion of lymphocytes or tumor cells to the lymphatic endothelium and lymphangiogenesis.

Tumor is still an unresolved health problem for human.The main obstacle for tumor cure is that precise diagnosis of tumor at the early stage can't be realized,which leads to the mortality increased year by year due to missing the best time of the treatment.Furthermore,lack of targeted ability and serious side effects of the medicine used for tumor treatment also limit the clinical application.Therefore,it is urgent to design and develop a novel targeted drug delivery vehicle.Recent studies show that targeted nanomaterials are superior on tumor-specific diagnosis and target therapy.This review will highlight the current advances in the targeted nanomaterials effect on tumor diagnosis and treatment in recent years and prospect the foreground of future development.

Objective To investigate the morphological characteristics of the autophagic structures in macrophages after phagocytosis of apoptotic lymphocytes and to explore the effects of autophagy on clearance of the apoptotic cells by macrophages. Methods Apoptosis of lymphocytes was induced with cyclophosphamide.The morphological changes of macrophages phagocytizing the apoptotic cells were viewed with a scanning electron microscope.The structural features of the autophagosome precursors,autophagosomes and autophagolysosomes in macrophages were examined with a transmission electron microscope,and the cross-section areas of the autophagic structures were measured with an image analyzer.The autophagosomes of macrophages were labeled with monodansylcaolaverine(MDC) staining and quantitated using laser scanning confocal microscopy.Results Macrophages actively phagocytized the apoptotic lymphocytes,apoptotic nuclei,apoptotic bodies and other cell debris to form heterophagosomes.When compared with the control group,numbers of autophagic cells and autophagosomes in these cells increase in the group of macrophages that engulfed the apoptotic cells.In addition,the ratios of the cross-sectional areas of the autophagic structures to that of the cytoplasm of the macrophages were greater.There were also apoptotic bodies or other cell debris in many the autophagosomes,and these autophagosomes were large and near the cell membrane.Autophagosomes containing the whole apoptotic cell or apoptotic nucleus were not observed.Conclusion The autophagic abilities of macrophages were significantly enhanced when the cells removed the apoptotic lymphocytes.Autophagy also plays an important direct or indirect roles in clearance of the apoptotic lymphocytes by macrophages.

Objective To investigate effects of endothelial adhesion molecule platelet endothelial cell odhesion molecule-1(PECAM-1),intercellular adhesion molecule-3(ICAM-3) and CD44 on lymphangiogenesis. Methods The lymphatic endothelial cells of the canine thoracic ducts were isolated and incubated.PECAM-1,ICAM-3 and CD44 on the lymphatic endothelial cells were labeled and visualized under fluorescence microscope and confocal laser scanning microscope.The endothelial cells stimulated by TNF-? or LPS were treated with the blocking antibodies against PECAM-1, ICAM-3 and CD44 respectively.The proliferation and migration of cells were determined with cell counting. The three-dimensional collagen gel model of lymphangiogenesis was prepared, and then the tube formation was viewed. The total length and area of the tubes formed from lymphatic endothelial cells were measured and their characteristics were examined with a transmission electron microscope. Results PECAM-1, ICAM-3 and CD44 were expressed on the lymphatic endothelial cells. In the control, TNF-? and LPS groups, the percentage of the migrated cells decreased and the total length and area of the tubes reduced after PECAM-1, ICAM-3 and CD44 of the cells were blocked respectively. The proliferated cells reduced after PECAM-1 or CD44 of the cells were blocked. However, there were no significant changes in the numbers of proliferated cells when ICAM-3 of the cells was blocked. In the semi-ultrathin and ultrathin sections, the tube structures formed by lymphatic endothelial cells were similar to lymphatic capillary.Conclusion PECAM-1, ICAM-3 and CD44 are expressed on the cultured lymphatic endothelial cells. These adhesion molecules are involved in lymphangiogenesis that includes proliferation, migration and tube formation of endothelial cells.

Objective To examine the distribution of HA in monocytes and expression of CD44 and ICAM-1 on the monocytes and effects of HA and HA receptors on adhesion and migration of the monocytes. Methods Canine peripheral blood monocytes were isolated by non-continuous density centrifugation with percoll solution.HA on the cell surface,intracellular HA,CD44 and ICAM-l of the monocytes were labeled with immunofluorescence.Effects of HA on the cell surface,substrate HA and free HA on adhesion and migration of the monocytes were examined by digesting HA on the cell surface with HAase,coating the bottom of the dishes and the membrane of the cell culture inserts with HA and adding HA into the medium.Regulating effects of HA receptors on adhesion and migration of the monocytes were investigated after blockade of the receptors. Results There was HA on the surface and in the cytoplasm of the monocytes.CD44 and ICAM-1 were expressed on the monocytes.The adhered and migrated monocytes decreased after HA on the cell surface was digested with HAase.The numbers of the adhered and migrated monocytes were greater when the cells were incubated on HA-coated dishes and membranes of the cell culture inserts.The adhered and migrated monocytes decreased after HA was added into the medium.When CD44 was blocked with antibody,the adhered and migrated monocytes decreased.There were not significant changes in the numbers of the adhered and migrated monocytes after ICAM-1 was blocked with antibody.Conclusion There is HA on the surface and in the cytoplasm of monocytes.HA on the cell surface is essential for adhesion and migration of the monocytes.HA substrate promotes adhesion and migration of the monocytes,while free HA reduces adhesion and migration of the cells.CD44 mediates HA-induced adhesion and migration of the monocytes.