| Infection and bleeding are two major lethal reasons for acute leukemia. Among many reasons for bleeding, disseminated introvascular coagulation (DIG) is the most dangerous, which often occurred in severe infection, especially, in septic shock. The patients developed multiple organ function failure, DIG and finally died. Most clinical studies suggested plasma fibronectin (FN) concentration might be a prognostic sign in the cases of septic shock complicated DIG, and a low plasma concentration of FN might define a group of septic patients with a high risk of developing DIG. Clinical trial suggested that reversal of plasma FN deficiency by infusion of FN-rich plasma cryoprecipitate resulted in a marked improvement in cardiopulmonary function in septic patients. Barkagan reported that FN-rich cryoprecipitate had a curative effect in 106 septic patients accompanied by DIG. Brodin reported that plasma cryoprecipitate infusion normalized the FN concentration of 28 patients with severe infection, and no patient had clinically overt DIG. These datasuggested that those patients with severe infection and DIG could be benefit from purified FN or cryoprecipitate therapy.Fibronectin (FN) is known to be a large multifunctional glycoprotein with binding sites for many substances, including heparin, collagen, fibrinogen, fibrin and integrin cell surface receptors. It plays an important role in a variety of cellular process, including tissue repair, blood clotting and cell migration/adhesion. FN interacts with cells by means of a cell-binding domain that contains RODS sequence, which is located in the 10th type III repeats of FN,and it has been shown to be a key attachment site for a variety of integrin cell surface receptors.Besides the RODS sequence, the synergy region in the 9th type III repeats have been shown to be the most important for interaction with FN receptors.EHlA,a spliced region of FN, known as connecting segment I (CS-I),is the second binding domain that also support cell adhesion.However, it is uncertain for the effect of FN on endotoxin and DIG, and no report about FN and its related polypeptide in treatment of sepsis and DIG has been found. In the present study, we examined the effect of FN on sepsis and DIG in mouse model of sepsis and rat model of DIG. Furthermore, we constructed a plasmid which carried a cDNA fragment coding for Ile 1363-Tyr 1725 of human FN,containing ROD, EIIIA and the synergy region in the 9th type III repeats,and then we induced expression of recombinant FN cell-binding domain polypeptide (rhFN55) in E.coli. By purification andidentification, the high pure and specific rhFN55 was achieved. These data provide a fundamental basis for FN and its related polypeptide as a supplementary therapy in sepsis and DIG. The subjects of thesis were focused on the following aspects:1. FN in the treatment of sepsis and DIG in an in vivo model.(1) Purification of FN from plasma was achieved by gelatin-sepharose affinity chromatography; the purified product was analyzed by SDS-PAGE and Western blot, and then was quantified by ELISA.(2) The effects of FN in the treatment of endotoxemia mice sensitized by D-galactosamine (GalN) were detected by histopathology, DNA fragmentation, electronmicroscopy, RT-PCR, ELISA.(3) The effects of FN in the treatment of rat DIG induced by LPS were detected by blood cell counter cytometry,semiautomatic hematology analyzer, histopathology and ELISA.2. rhFN55 in the treatment of sepsis and DIG in vivo model.(1) By gene recombinant technique, the plasmid was constructed to express rhFN55, and the expression product was analyzed by SDS-PAGE and Western blot.(2) By biotin-affmity chromatography, rhFN55 was achieved; the purified product was analyzed by SDS-PAGE and then quantified by Bradford reagent.(3) The cell adhesion activities of rhFN55 and commercial FNpolypeptide (G4393 is the synthetic peptide containing ROD sequence that is major cell-binding site; F3667 is an adhesion -promoting...
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