The Effect Of Recombinant N-terminal And C-terminal Heparin-binding Domain Polypeptides Of Fibronectin On Septicemia Induced By The Infection Of Escherichia Coli In Mice

Clinically, sepsis is an acute severe syndrome. Although there are many antibiotics which have high performance to produce and more and more consummate organ support and surveille technique, the mortality is still high. Thus, people are always developing new effective drugs to cure sepsis. Fibronectin (FN) is known to be a large multifunction glycoprotein with binding sites for many substances, including heparin, collagen, fibrinogen, fibrin and integrin receptors. It is involved in a variety of cellular processes, including tissue repair, blood clotting and cell adhesion or migration. Experimental studies suggested FN purified from plasma have the effect on sepsis, clinically, cold sediment of FN had achieved good effect on sepsis . But pure FN is not yet used to cure desease in clinical. FN can been purified from plasma, but this method might transmit infectious diseases,such as HIV and waste the blood source. Meanwhile because of the high molecular weight (450kDa) of FN, it is difficult to express the whole molecular of FN by genetic engineering. Because there are many functional structural domain such as heparin-binding domain,cell-binding domain,fibri-binding domain and so on , the function of FN can been came true through these structural domain. Thus, it is necessary to express the related function polypeptides and study the function of recombinant polypeptides on sepsis.Previous studies of our experiment group had expressed and purified the recombinant cell- binding domain polypeptide of FN(rhFN55 polypeptide), and confirmed it had the effect on mice with endotoxemia induced by D-Galactosamine sensitizing and DIC induced by endotoxin;We also had constructed the recombinant yeast expressed vector with the N-terminal and C-terminal heparin-binding domain polypeptides of FN, and filtered high expressive bacterial strain, further expressed and purified N-terminal and C-terminal heparin-binding domain polypeptides( FNNHBDPP and FNCHBDPP, and studied the function of recombinant polypeptides on mice with endotoxemia induced by D-Galactosamine sensitizing, and confirmed that both FNNHBDPP and FNCHBDPP had the protective effect to the model of mice. Yet , though endotoxin plays a predominant role in mice with endotoxemia induced by D-Galactosamine sensitizing and mice with septicemia induced by the infection of Gram-negative bacteri. But, the latter is also related to Gram-negative bacteria itself besides endotoxin. Therefore the pathogenesis of septicemia induced by the infection of gram-negative bacteria is much closer to clinical sepsis. In order to understand preferably the effects of FNNHBDPP and FNCHBDPP on mice model of different septicemia, in this study, we will further study on the effects of the two polypeptides on the mice with septicemia induced by the infection of E.coli.The effects of FNNHBDPP and FNCHBDPP on this septicemia model is unclear.The subjects of thesis were focused on the following aspects:1.The preparation of two heparin-binding domain polypeptides(FNNHBDPP and FNCHBDPP).FNNHBDPP and FNCHBDPP were obtained by expression and preparation in Pichia yeast expression system.2.The effects of FNNHBDPP,FNCHBDPP on septicemia induced by the infection of E.coli in mice.(1) The establishment of mice septicemia model induced by the infection of E.coli. We strengthened toxicity of the E.coli strain in vivo of mice, and incubated the Ecoli for 18 hours, whose toxicity had been enhanced, then diluted the bacterium fluid to different kinds of concentration using 5% high activity dry yeast, feeled the minimum lethal dose(MLD. We diluted bacterium fluid to final concentration that we need, injected 0.5ml 1MLD to intra-peritoneal, the model of septicemia had been finished. (2) The observation of the effect of using FNNHBDPP,FNCHBDPP alone and combing Ceftriaxone whose doses is maximum noneffective①Experimental group;We used two doses of FNNHBDPP namely 20mg/kg,40mg/kg,two doses of FNNHBDPP namely 40mg/kg,60mg/kg to cure the sepsis mice, groped the effective dose is 40mg/kg,60mg/kg respectively. On the basis of the result, we set up experimental group as follows: The normal group, the physiological saline control group, 20mg/kg (maximum noneffective dose) Ceftriaxone group, FNNHBDPP group, FNCHBDPP group, the group of FNNHBDPP combining Ceftriaxone, the group of FNCHBDPP combining Ceftriaxone.②The observation of 72 hours survival rate after the injection of bacteria in mice ;③The detection of serum ALT,AST,TBIL,LDH,BUN,Cr,CK,CK-MB;④The detection of plasma TNF-α,IL-1β,IL-6 by ELISA;⑤the observation of Liver tissue pathology and ultrastructure .The major results and conclusions were obtained as follows:1. The mice septicemia model was established.2. The result of 72 hours survival rates showed that the survival rates of the physiological saline control group was 5%, that of FNNHBDPP group and FNCHBDPP group were 22.5% and 25.3% respectively, by X2 test, compared to the control group P < 0.05. The survival rates of FNNHBDPP combing Ceftriaxone and FNCHBDPP combing Ceftriaxone were 42.7% and 43.5%, by X2 test, compared to the control group P < 0.01. FNNHBDPP group and the group of FNNHBDPP combining Ceftriaxone, by statistical analysis two are no significant differences. So did FNNHBDPP group and the group of FNNHBDPP combining Ceftriaxone. In addition, we comepared different administer drug times based on the same doses of two polypeptides, it showed that the changes of survival rates were unconspicuous if only use FNNHBDPP 40mg/kg or FNCHBPP 60mg/kg for one time or two times. But if increasing the times of administration of FNNHBDPP and FNCHBDPP such as for three times or four times the survival rates of FNNHBDPP-treated group were 20%, 22.5%, by X2 inspection, two are no significance and FNNHBDPP-treated group were 21.7%,25.3%, by statistical analysis, two are aslo no significant differences.3. By detecting the levels of serum TBIL,AST,ALT,LDH,BUN,Cr,CK,CK-MB, it indicated that both FNNHBDPP and FNCHCDPP can decrease the levels of serum TBIL,AST,ALT,LDH,CK,CK-MB and improve the liver function and myocardial enzymes, and it showed better therapeutic effect by combining low dose Ceftriaxone.4. The levels of TNF-α,IL-1β,IL-6 in plasa determined by ELISA in the control group were higher than in the normal control and the treatment group of FNNHBDPP and FNCHBDPP, and it showed better therapeutic effect by combining low dose Ceftriaxone.5. Through the observation of pathology and ultrastructure of organs by light microscope and electron microscopy, the results showed that FNNHBDPP and FNCHBDPP can lighten the damage of the liver and inhibit liver cell necrosis. The results also showed that two polypeptides showed better therapeutic effect by combining low dose Ceftriaxone.6. Conclusion: the experimental results suggested that FNNHBDPP and FNCHBDPP can improve the survival rates of the mice model. Moreover if using low dose(20mg/kg) Ceftriaxone meanwhile they will show better therapeutic effect. FNNHBDPP and FNCHBDPP lighten biological effects and damages of the liver and other organs caused by these inflammatory factor possibly through inhibiting the production of TNF-α,IL-1β,IL-6 etc. The therapeutic effects of FNNHBDPP and FNCHBDPP have no significant differences.