The micropipette aspiration technique was improved by selecting the uniform cells in round shape (UCR) from a number of adhered cells on the testing surface so as to measure the adhesion forces during the initial adhesion stage. Silk fibroin film and silk fibroin/fucoidan film were used as testing substrates. The testing results revealed that the modified micropipette method was more rapid for testing. The tendency of cell adhesion force on different materials surface under different conditions remained unchanged. The testing results showed good reproducibility. It was easy to distinguish small differences in the adhesiveness of endothelial cell on the silk fibroin based materials, thus exhibiting the high sensitivity of the improved method.
Cell Adhesion ; physiology ; Cells, Cultured ; Endothelial Cells ; cytology ; Fibroins ; Humans ; Umbilical Veins ; cytology

OBJECTIVETo investigate the variety of proliferating ability of umbilical endothelia (UE) transfected by plasmid pBABE-HYGR-hTERT.

METHODSUE was identified from two aspects: morphology and CD34 labeling technique. The plasmid was obtained and identified by alkali splitting and gel electrophoresis. Liposomes were used to transfect UE. RT-PCR based telomeric repeat amplification protocol (TRAP) assay was used to measure the telomerase activity of endothelia.

RESULTSUE arranged as "cobblestone" and were positive of CD34 labeling. Endothelia transfected by pBABE-HYGR-hTERT(HC) had an raised absorbance of 0.889. The shape of growth curve of HC was similar to UE. But the absorbance of MTT test and the amount of HC were prior to UE at every measuring time and the amount of HC increased four times within 8 days (P < 0.05).

CONCLUSIONThe transfection of pBABE-HYGRO-hTERT had greatly improved the proliferating abilities and activated the telomerase of UE.

Catalytic Domain ; genetics ; Cells, Cultured ; Endothelium, Vascular ; cytology ; Humans ; Telomerase ; genetics ; Transfection ; Umbilical Veins ; cytology

This study was aimed to investigate whether mesenchymal stem cells (MSCs) existed in human umbilical cord vein and to establish the methods of isolation and expansion in vitro. The MSCs derived for perfusion of umbilical cord vein (UVMSCs) were collected after parturition of Healthy pregnant women. The morphology of MSCs and their differentiation potential into osteoblast, adipocyte and chondrocyte were observed the phenotype and cell cycle of MSCs were determined by using flow cytometry. The result showed that the mesenchymal stem cells separated from umbilical cord vein gave rise to a population of adherent cells with a typical fibroblast-like morphology. Similarly to bone marrow-derived MSCs, they highly expressed CD29, HLA-ABC, CD166, CD105, CD73 and CD44, and were negative for any hematopoietic and endothelial markers (CD45, CD34, CD14 and CD144). Functionally, they could differentiate into the osteoblast, adipocyte and chondrocyte. It is concluded that MSCs exist in the human umbilical cord vein perfusion. Their read amplification in vitro contribute to clinical applications for cell therapy and tissue engineering.
Cell Differentiation ; Cell Separation ; methods ; Cells, Cultured ; Female ; Fetal Blood ; cytology ; Humans ; Mesenchymal Stromal Cells ; cytology ; Umbilical Veins ; cytology

This study was aimed to compare the differences of adhesion properties of endothelial cells (EC) from arteries (AEC), veins (VEC) and capillaries (MVEC) under shear stress condition, and to explore whether they can get the same adhesive ability as graft in similar shear stress conditions. With mended parallel plate flow apparatus and peristalsis pump providing fluid shear stress used, endothelial culture models were established in vitro with the same environmental factors as steady culture. To compare the adhesion among three kinds of endothelial cells under dynamic condition and static condition, the dynamic change of cytoskeletal actin filaments and the effects of different adhesive proteins coated on the adhesion of EC to the glass were studied. The cultured endothelial cells under flow conditions were extended and arranged along the direction of flow. The adhesive ability from high to low under static condition were AEC, MVEC and VEC (VEC compared with AEC or MVEC, P < 0.05), sequentially. The adhesion of endothelial cells from variety sources under dynamic culture condition was significantly increased than that of the static groups. The ratio of cell retention was not significantly different between AEC and MVEC. But VEC was significantly different (P < 0.05) compared with AEC or MVEC. The actin filaments (F-actin) were bundled together and arranged along the direction of flow after fluid culture. Dense peripheral band (DPB) gradually disappeared and distinct stress fibers were formed, which even interconnected to form a whole in the MVEC. The adhesion of AEC, VEC and MVEC under shear stress conditions are more significantly increased than those under the static culture conditions, and the MVEC can achieve the same adhesion as AEC.
Arteries ; cytology ; Capillaries ; cytology ; Cell Adhesion ; Cell Culture Techniques ; methods ; Cells, Cultured ; Cytoskeleton ; physiology ; Endothelial Cells ; cytology ; physiology ; Humans ; Shear Strength ; Stress, Mechanical ; Veins ; cytology

PURPOSE: This study was performed to examine the vascular network of the human iris using flat preparation. METHODS: The ciliary body-iris structures were separated from human eyeballs, and a portion of the irises were treated with trypsin to remove the pigment granules. These iris tissues were unfolded and placed onto glass slides using flat preparation, and the vascular network of each iris was examined by fluorescein microscopy. The ciliary body-iris structures separated from the remaining eyes were stained with hematoxylin-eosin without trypsin treatment and were examined by light microscopy. RESULTS: The long posterior ciliary artery formed several branches before entering the iris root, and such branches formed the major arterial circle of the iris with diverse diameters in the vicinity of the iris root and the ciliary process. In the pupillary margin, the iris vasculature network formed a cone shape and then formed an arcade by connecting to adjacent vasculatures. In the vicinity of the collarette, the iris vasculature network formed the minor arterial circle of the iris with diverse diameters perpendicular to the arcade of the iris network located in the pupillary margin. In the pupillary margin, the capillaries were somewhat thick and connected to the irregular traveling iris vein. CONCLUSIONS: The above findings explain the human iris vascular network and provide a theoretical basis for the sectoral filling of the iris vasculature seen in fluorescein iris angiography.
Cadaver ; Cytological Techniques/*methods ; Humans ; Infant ; Infant, Newborn ; Iris/*blood supply ; Microscopy, Fluorescence ; Ophthalmic Artery/*cytology ; Veins/*cytology

OBJECTIVETo design and construct the inducible expression vector of endothelial-overexpressed lipopolysaccharide-associated factor 1 (EOLA1), in order to establish EOLA1 compelling expression model, and to observe the effects of EOLA1 compelling expression on the proliferation of ECV304 cells.

METHODSInducible overexpression vector pOPRSV I-EOLA1 was constructed by amplifying the open reading fragment of EOLA1 and subcloning it into the Not I site and Xho I site of pOPRSV I vector. After sequencing, the pOPRSV I-EOLA1 recombinant vector and pCMVLac I vector were co-transfected into ECV304 cells. The cells resistant to G418 and hygromycin were screened by G418 and hygromycin, so that stable transfected cell strain was obtained. The growth curve of cells with or without isopropyl-beta-D-thiogalactoside (IPTG) induction were graphed with cell counting.

RESULTSThe inducible overexpressed EOLA1 vector was constructed successfully. The proliferation of the cells with EOLA1 compelling expression after induction of IPTG (44 +/- 17) x 10(4) was significantly higher than that without IPTG induction (27 +/- 11) x 10(4), (P < 0.01).

CONCLUSIONCompelling expression of EOLA1 protein can enhance the proliferation of ECV304 cell.

Cell Line ; Cell Proliferation ; Endothelial Cells ; cytology ; Gene Expression ; Humans ; Lipopolysaccharides ; Membrane Proteins ; genetics ; Transfection ; Umbilical Veins ; cytology

Fluid shear stress plays an important role in many physiological and pathological processes of the cardiovascular system. Being constantly exposed to mechanical shear stress, vascular endothelial cells can sense the changes of blood flow forces and regulate vascular structure and function. Previous studies demonstrated that IL-8 mRNA expression in endothelial cells was modulated by fluid shear stress. To identify the effect of fluid shear stress on IL-8 protein production of human umbilical vein endothelial cells (HUVECs), we employed quantitative sandwich enzyme-linked immunosorbent assay (ELISA) to measure the IL-8 protein. It was found that the HUVECs not treated with fluid shear stress secreted very little IL-8 in culture media. However, after 1 hour of exposure to shear stress, the secretion of IL-8 increased; at 5 hours of exposure, the seceretion reached the summit; at 8 hours of exposure, the secretion of IL-8 decreased and then remained at a constant level till the end (12 hours) of the experiment. The increase of IL-8 secretion induced by shear stress was time-dependent. The biphasic response of IL-8 protein production was found in experiments in which the shear stress applied was 2.09 dyne/cm2, 4.61 dyne/cm2, and 6.19 dyne/cm2. The IL-8 protein production in response to shear stress was very similar to the IL-8 gene expression in response to shear stress, and had the obvious delay. The induction of IL-8 protein production by fluid shear stress is probably due to the gene expression. This in vitro study demonstrates that the production of IL-8 can be regulated by fluid shear stress. Fluid shear stress induces a biphasic response of human HUVECs' production of IL-8 protein. These observations suggest that the process of the fluid shear stress induced HUVECs' production of IL-8 may play an important role in the genesis and development of both inflammation and atherosclerosis.
Cells, Cultured ; Endothelial Cells ; metabolism ; Endothelium, Vascular ; cytology ; Humans ; Interleukin-8 ; biosynthesis ; Stress, Mechanical ; Umbilical Veins ; cytology

Extensive monocyte recruitment is an early phenomenon associated with the development of atherosclerotic lesion. Although the molecular mechanisms are not completely understood, monocyte recruitment into these early lesions may involve changes in endothelial adhesion for monocyte, in which adhesion molecules expressed by endothelial cell play an active role. In vivo, the function of endothelial cells is not only affected by the chemical factors, but also by the mechanical factors. The purpose of this article was to investigate the induction of adhesion molecules expression by synergistic effects of Lysophosphatidylcholine (Lyso-PC) and shear stress in cultured human umbilical vein endothelial cells (HUVEC). The surface expression of ICAM-1, VCAM-1 and E-selectin on HUVEC induced by Lyso-PC(30 micrograms/ml) and shear stress(2.23, 4.20 dyne/cm2) were analyzed using flow cytometry. The results showed that: Compared with what were simultaneously exposed to shear stress and Lyso-PC, activating the cells with Lyso-PC prior to shear stress, or pre-conditioning the cells exposed shear stress prior to Lyso-PC incubation, a significantly higher expression of ICAM-1 and VCAM-1(P < 0.05) was resulted. HUVEC were exposed to shear stress and Lyso-PC at the same time or treated with each agonist alone, E-selectin expression was not significantly different from the control group. However, a sequential action of the two stimuli significantly increased E-selectin expression(P < 0.05). We concluded that: a sequential action of the shear stress and Lyso-PC induced an even greater expression of ICAM-1 and VCAM-1, thus it could be understood that the flows-hear stress in combination with endothelial activated by chemical factors may increase the ability of endothelial cells to recruit leukocytes even under the mechanical environment unfavorable for cell adhesion.
Cell Adhesion Molecules ; biosynthesis ; Cells, Cultured ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Humans ; Lysophosphatidylcholines ; pharmacology ; Stress, Mechanical ; Umbilical Veins ; cytology

Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Endothelium, Vascular ; cytology ; metabolism ; Gene Expression Profiling ; Humans ; Ouabain ; pharmacology ; Umbilical Veins ; cytology

OBJECTIVETo observe the effect of Porphyromonas gingivalis (Pg) on the production of nitric oxide (NO) in cultured human umbilical vein endothelial cells (HUVEC), and to investigate the pathway of damaging endothelial function by Pg.

METHODSPg ATCC33277 was cultured in anaerobic jar, and HUVEC was treated with various concentrations of Pg ATCC33277 at multiplicity of infection (MOI) of 1:10, 1:100 and 1:1000 for 4, 8, 12, 24 h respectively. The cells supernatants were collected and stored at -70 degrees C and NO concentration in the cells supernatants was measured by nitrate reductase assay.

RESULTSWithin 24 h, Pg at MOI of 1:10 and 1:100 stimulated the release of nitric oxide in cultured HUVEC. Within 12 h, Pg at an MOI of 1:1000 group increased NO production, and NO decreased at 24 h.

CONCLUSIONSPg has an effect on the production of NO. Low concentrations of Pg stimulated release of nitric oxide in endothelial cells but high concentrations can decrease the release of NO.

Cells, Cultured ; Endothelial Cells ; metabolism ; Humans ; Nitric Oxide ; biosynthesis ; Porphyromonas gingivalis ; isolation & purification ; Umbilical Veins ; cytology