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Vascular endothelial cells have very important physiological functions, mainly reflected in: 1. Constitute a natural barrier, and maintenance of intimal smooth, prevent platelet and leukocyte adhesion and harmful substances invade the blood vessel wall; 2 form a permeability barrier, regulating the exchange of material and active transport function; 3. involved in thrombosis and hemostasis, maintain homeostasis, including tissue plasminogen activator (t-PA), plasminogen activator inhibitor -1 (PAI-1). Many pathological states such as serious infections, DIC and so involve dysfunction of the vascular endothelial cells, which in turn exacerbate the pathological process, creating a vicious cycle. Therefore, how to restore the form and function of the pathological state of imbalance in vascular endothelial cells have been the focus of academic research. Fibronectin (Fibronectin, FN) is a widely present in the extracellular fluid, the cell surface and connective tissue, etc. of the glycoprotein. , So there have been opsonic α2 surface binding protein, cell surface proteins, and cell adhesion molecules known to play an important role in the anti-infective maintain microvascular integrity and permeability. The known the FN mainly divided into two kinds of cells and plasma cell type FN mainly by endothelial cells and fibroblasts, for the connection between endothelial cells and endothelial cell adhesion to endothelial lower collagen fibers play an important role, which ensure microvascular completeness. Therefore, the study of exogenous FN certain pathological factors caused the repair of the vascular endothelial cell injury is valuable, given the existence separated from plasma FN plasma lack of resources and the possible spread of blood-borne infectious diseases, also due the FN molecular weight 450KD, the whole molecule expression and there is a technical problem. Therefore expression of FN function polypeptide using genetic engineering techniques and to study its function, instead of the FN can be said to be a viable option. TNF-α is an important inflammatory mediators, TNF-α increased infections and other pathological cases, our previous studies have shown that the FN and the heparin-binding domain polypeptide can prevent the occurrence of lipopolysaccharide endotoxin in mice DIC mechanism involving Inhibition of the expression of TNF-α. Therefore, based on the basis of the preliminary study, the aim of this experiment is to establish a mouse model of endotoxemia in TNF-α vascular endothelial cell injury model and galactosamine sensitizing by in vitro and in vivo experiments, research FN and heparin domain of polypeptide FNNHBSPP, FNCHBSPP vascular endothelial cells. Human umbilical vein endothelial cells cultured and identified using the enzyme digestion method in cultured human umbilical vein endothelial cells; using immunohistochemical method detection of factor Ⅷ identification endothelial cells. 2.FN and two types of heparin binding domain polypeptide FNNHBSPP and the FNCHBSPP preparation with gelatin affinity chromatography from fresh human plasma separation FN; were identified by SDS-polyacrylamide gel electrophoresis and immunoblotting (Western blot) FN purity and specificity The nature; two heparin binding domain polypeptide FNNHBSPP expression and preparation of the yeast expression vector FNCHBSPP self-prepared by the Institute. 3 TNF-α injury in human umbilical vein endothelial cell model 3.1 Experimental groups: (1) blank control group: without the normal growth of the TNF-α treatment HUVECs; (2) different concentrations of TNF-α treatment groups: respectively 5ng / ml and 10 ng / ml and 20 ng / mlTNF-α role 18h; (3) different times of TNF-α treatment group: 10 ng / mlTNF-α role 6h, 12h, 18h, 24h. 3.2 in each group were measured by ELISA endothelial cell culture supernatant of PAI-1, tPA, sICAM-1 content. Research FN and two heparin binding domain polypeptide FNNHBSPP, 4.1 impact of FNCHBSPP the endothelial cells of the role of TNF-α experiment groups: (1) blank control group: without the normal growth of the TNF-α treatment, HUVECs; (2) TNF-α action group: 10ng/mlTNF-α role 18h, joined with a polypeptide, the volume of the culture liquid 6h; (3 ) TNF-α FNNHBSPP role group: 10ng/ml TNF-α role 18h, added at a concentration of 600ug/ml of FNNHBSPP role 6h; (4) TNF-α FNCHBSPP role of: 10ng/mlTNF-α role 18h, added at a concentration of 600ug / ml of FNCHBSPP role 6h; (5) of TNF-α FN action group: 10ng/mlTNF-α role 18h, added in concentrations of 600ug/ml the FN effect 6h. 4.2 inverted microscope and transmission electron microscopy the FN of its two heparin binding domain polypeptide FNNHBSPP, morphology and ultrastructure 4.3 FNCHBSPP endothelial cells under the action of TNF-α injury groups endothelial cell culture supernatant of PAI were detected by ELISA -1, tPA, sICAM-1 levels 5. research FN its two heparin-binding domain polypeptide FNNHBSPP, FNCHBSPP 5.1 Establish internal endotoxemia mouse liver, vascular endothelial cells of the lung tissue galactosamine within sensitizing endotoxemia in a mouse model of application galactosamine sensitizing lipopolysaccharide establish a mouse model of endotoxemia, that is, to mice by intraperitoneal injection of galactosamine (400mg/kg) and endotoxin (100ug/kg,). The 5.2 experimental groups: normal group, endotoxemia group the FN action group, FNNHBSPP action group and FNCHBSPP role group. Half an hour before the injection galactosamine and endotoxin from the tail vein injection the FN, FNNHBSPP, FNCHBSPP (20mg/kg), endotoxemia group to an equal volume of normal saline. 5.3 pathological morphology in each experimental group of mice liver, intraperitoneal injection of drugs after nine hours of vascular endothelial cells of the lung tissue, cervical dislocation, the mice were killed, take the mouse liver, the lung tissue, with 10% formalin-fixed, paraffin embedded, sliced, routine HE staining, pathological morphological observation. 5.4 immunohistochemistry assay liver, vascular endothelial cells of the lung tissue fibers connected proteins (FN) expression made the following findings and conclusions: 1. Establish stable passaged human umbilical vein endothelial cell culture system. 2 established a model of endothelial cells in TNF-α, the role of different concentrations of TNF-α (5,10,20 ng / ml) in HUVECs18h culture medium in PAI-1, sICAM-1 concentration compared with the blank control group groups had increased (p <0.01), of which the most obvious 10ng/ml TNF-α role; role of TNF-α with the same concentration of 10ng/ml 6,12,18,24 h, compared with the control group, PAI-1, sICAM-1 concentration were significantly increased (p <0.01), the most significant role in the 18hTNF-α; that of tPA groups was no significant difference (p> 0.05). 3.FN its The heparin binding domain polypeptide FNNHBSPP, the FNCHBSPP role group endothelial cells, cultured supernatants of PAI-1 and sICAM-1 concentration than the TNF-α group was significantly lower (p <0.01), FN and two The polypeptide role no significant difference (p> 0.05). 4. Establish a mouse model of endotoxemia, I FN and polypeptide FNNHBSPP, the group of mice liver FNCHBSPP role, the endothelial cells of the lung tissue lesions significantly reduced FN expression of the vascular endothelial cells than mice with endotoxemia enhanced. Conclusion: FN and its polypeptide FNNHBSPP FNCHBSPP TNF-α and vascular endothelial cell damage caused by sepsis has a protective effect; FN and its two heparin-binding domain polypeptide role in this regard was no significant difference.
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