Effects Of Fibronectin And Its Recombinant Heparin-binding Site Polypeptides On Vascular Endothelial Cells

Vascular endothelial cells are an interface between blood vessel walls and blood flow, and play important roles in physiological functions.1.Constitute a natural barrier to maintain endometrial vascular smooth, preventing platelet and leukocyte adhesion and harmful substances invasive vascular wall.2.Permeability barrier formed to regulate the exchange of material and active transport function .3.Thrombosis and hemostasis involved in the process, and maintain dynamic equilibrium ,which is including tissue plasminogen activator, plasminogen activator inhibitor-1 (PAI-1). Many pathological conditions such as severe infection, DIC, are involved in vascular endothelial cells function, and then aggravate the pathological process, forming a vicious cycle. Fibronectin (FN) is a glycoprotein which is presence in the extracellular matrix, plasma and other body fluids .As an important adhesion molecule with several integrin receptor binding, FN participates in a number of important physiological and pathological processes including cell adhesion, migration and extracellular matrix assembly, embryonic differentiation, wound healing, immune and inflammatory. And various diseases are closely linked. FN not only has a function anti-infective and but also maintain the integrity and permeability of microvascular. It is also called"rationalα2 surface-binding protein", "cell surface protein" and "cell adhesion molecules." FN is known mainly divided into two types: cellular and plasma-fibronectin. cFN is secreted by endothelial cells and fibroblasts. It has play an important role over the endothelial cells surface and endothelial cells attached to the subendothelial layer collagen fibers .So cFN ensure the integrity of microvascular .It is great value to study how exogenous FN repair vascular endothelial cells on certain pathological .There are still some problems accompanying with extracting FN from human plasma ,for example lack of blood resource ,spread of blood-borne infectious disease , big molecular weight and the technical problems to expression whole molecule . Therefore using genetic engineering technology express the peptide functional areas of FN and study their functions. It is a practical ways using recombinant polypeptides to replace FN. TNF-αis an important inflammatory mediators. In some pathological cases, such as infection, there is an increased TNF-αlevel. Our previous studies showed that FN and recombinant heparin-binding domain peptide can control endotoxin lipopolysaccharide mice in the DIC and the mechanism involving the suppression of TNF-αexpression. Therefore the main purpose was to study the influence of FN and its polypeptides on vascular endothelial cells in vitro and in vivo experiments.The subjects of thesis were focused on the following aspects:1. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunohistochemistry;2. FN and its recombinant heparin-binding site polypeptides preparation. Purification of FN from plasma was achieved by gelatin-sepharose affinity chromatography. The purifed product was analyzed by SDS-PAGE and western blot, and then was quantified by ELISA. The high pure and specific FNNHBD and FNCHBD were obtained by expression and preparation of recombinant FN heparin-binding domain polypeptide in pichia expression system;3. Established a TNF-αdamage endothelial cells model.3.1 Experimental group :(1) the first group of HUVECs was blank group;(2) the second group were treated with 5ng/ml,10 ng/ml or 20 ng/ml TNF-αfor 18h;(3) the third group were treated with 10 ng/ml TNF-αfor 6h,12h,18h or 24h respectively.3.2 PAI-1, tPA, sICAM-1 levels in the endothelial cells culture supernatant were detected by ELISA.4. We investigate the effect of FN and the two polypeptides on HUVECs following treatment with TNF-α.4.1 Experimental group :(1) the first group of HUVECs was blank group;(2) the second group were treated with 10 ng/ml TNF-αfor 18h,and then mediam which has the same volume as peptides was added;(3) the third group were treated with 10ng/ml TNF-αfor 18h,and then 600 ug/ml FNNHBSPP was added;(4) the fourth group were treated with 10ng/ml TNF-αfor 18h,and then 600 ug/ml FNCHBSPP was added;(5) the last group were treated with 10ng/ml TNF-αfor 18h,and then 600 ug/ml FN was added.4.2 Observe the endothelial cells morphology and ultrastructure using an inverted microscope and transmission electron microscopy.4.3 PAI-1, tPA, sICAM-1 levels in the endothelial cells culture supernatant were detected by ELISA;5.Study Fibronectin and Recombinant Heparin-binding Site Polypeptides of Human Fibronectin' effects on sepsis mouse VECs.5.1 Establishment of sepsis mouse model.5.2 Experimental group : the normal group,the blank group,FN-treated group,FNNHBSPP-treated group and FNCHBSPP-treated group. We established the septic mouse model by the injuection of 100ug/kg lipopolysaccharide(LPS)and 400mg/kg D-Galactosamine(GalN) into the mice which is according to A.-H. Kwon,s and our test condiction. We indicated that FN,FNNHBSPP and FNCHBSPP administrated intravenously 30min before LPS/GalN injection at the base of 20mg/kg. The control group gave the volume saline.5.3 Pathological observation of the liver, lung vascular changes . 9h after intraperitoneal injection drug, the mice were killed by cervical dislocation. The mice liver and lung tissues fixed with 10% formalin, embedded in paraffin, slicing, conventional HE staining.5.4 Immunohistochemical detect the vascular endothelial cells fibronectin (FN) expression.The major results and conclusions were obtained as follows:1. Isolation and culure human umbilical vein endothelial cells;2. Established TNF-αdamage endothelial cells model.Our results shew that TNF-alpha increased the levels of PAI-1 and sICAM-1 in cultured human umbilical vein endothelial cells (HUVECs) compared HUVECs in normal conditions(p﹤0.01); And the maximum stimulatory effect of 10ng/ml of TNF-alpha was observed after 18h. There was no significant difference of tPA level(p﹥0.05);3. The levels of PAI-1 and sICAM-1 in cultured HUVECs which were treated with FN,FNNHBSPP or FNCHBSPP were decreased than the TNF-alpha -treated group; Also, there was no significant difference between the FN,FNNHBSPP and FNCHBSPP treated groups(p﹥0.05);4. We established the septic mouse model. The Vascular endothelial cells injury risks in septic mouse's liver and lung were reduced which were treated with FN and FNNHBSPP, FNCHBSPP. Also, the expression of FN in vascular endothelial cells was more than that of the septic mouse;5. Conclusion: FN and its polypeptides FNNHBSPP,FNCHBSPP had a protective effect on VECs which were damaged by TNF-αor LPS/GalN.Also, there was no significant difference between the FN,FNNHBSPP and FNCHBSPP treated groups.