| Atherosclerosis (AS) is the common pathological basis of many cardio and cerebral vascular diseases, and also one of the most frequent disease with serious threat to human health. Although many researchers proposed different theories about AS pathogenesis however, anyone of which could not alone elucidate the genesis and development of AS completely. In the recent years, lots of experimental researches at cellular and molecular levels indicated that AS resulted in a similar development pathway, i.e. chronic inflammatory pathological process. The development of AS was accompanied by some similar pathophysiological process, including hemoendothelial damage, oxidation and infiltration of lipid, the activation, adhension and infiltration of platelet and inflammatory cells, the immigration and proliferation of smooth muscle cells etc.. On the basis of the AS development characteristics, we consider whether the multi-targeted drug could be constructed and applied, and then the effect against AS could be better.Studies displayed that inflammation penetrated through all the process of AS genesis and development, which was a common basis of many pathophysiological changes. Thrombin and platelet as the targeting sites for many anti-coagulant drugs were the important factors involved in blood coagulation, while more and more researches indicated that thrombin and platelet accumulated at damaged site of vessel played an important promoting role for the inflammatory response of vessel wall. Because of the hyperfunction of local blood coagulation, a large amount of platelet, leukocyte and thrombin accumulated, the existence of which exacerbated the inflammatory reaction at the damaged blood vessel and then accelerate the development of atherosclerosis, therefore thrombin and platelet simultaneously participated in both blood coagulation and inflammation. In the late period of AS development, the patients were often accompanied by the symptoms of coronary syndrome, to which the anticoagulant and thrombolysis therapies were applied .Therefore, we designed and constructed a multifunctional fusion protein SRH to perform the anti -AS effect through its anticoagulation, anti-inflammation and thrombolysis activity.Natural staphylokinase (SAK) as a selective fibrinogen activator possessed some advantages, such as higher fibrinogen specificity, stronger action on platelet-enriched thrombus, lower side-effect, and cheaper price. The clinical trial evaluation of our rSAK has been completed, and the results indicated that its recanalization rate was significantly higher than that of r-tPA, thus showing a good perspective of clinical application. We designed a multifunctional fusion protein SRH by using the modified SAK as the backbone. The molecular structure of SRH consisted of three functional domains: the modified SAK molecule, RGD sequence, and dodecapeptide of hirudin. SAK displayed mainly the dissolution of fibrin, RGD sequence possessed inhibition effect on platelet aggregation, and the dodecapeptide of hirudin had anti-thrombin function.This study mainly includes the following four parts:1. Overlapping extension PCR methods were used to construct the recombinant gene encoding SRH fusion protein, which was inserted in the expression vector pBV220, and the resulted recombinant plasmid was used to transform the competent cells of E.coli BL21 to obtain an engineering bacteria strain for inducing expression of SRH. The results showed that SRH could be expressed in the soluble form by the engineering strain E.coli BL21/pBV220-SRH, and SRH could be purified after expression in large scale. The purity of SRH reached to 95% after two steps of purification with anion-exchange chromatography and gel filtration. The functions of different domains of the fusion molecule SRH were analyzed by using the lysis circle assay on fibrin agarose gel plate and chromogenic substrate method. The results showed that the thrombolytic activity of SRH was (1.3±0.15)×104 AU/mg, lower than that of SAK((4.1±0.12)×104 AU/mg). Binding ability analysis indicated that SRH was able to bind with thrombin, and further assay with fibrin clotting method showed SRH had stronger anti-coagulation function than that of SAK. Assay of SRH anti-platelet aggregation function displayed that SRH maintained the function of RGD anti-platelet aggregation. The above results demonstrated the fusion protein constructed in our research maintained the functions of three domains, possessing thrombolysis, inhibition of platelet aggregation and anti-thrombin.2. We utilized apoE-/-mouse as the animal model feeding with high fat diet to observe the progression of atherosclerosis after injection with SRH. H.E. staining of aorta sinus demonstrated the model group showed a serious lesion of AS, and AS plaque occupied 91% of the aorta lumen area. The group treated with SRH displayed different results: 39% of that for SRH 1mg/kg body weight, and 43% for SRH 0.125 mg/kg. These results suggested that the plaque lesion of AS in the SRH-treating group had tendency of attenuation.3. In order to elucidate the anti-atherosclerosis mechanism of SRH protein, the effect of SRH protein on HUVEC cell was tested. The results showed that SRH protein could down-regulate expression of AS-related genes including PAI, ICAM-1, VCAM-1, IL6, IL8, MCP-1 and TF. SRH also inhibited the secretion of IL6 and MCP-1.These results suggested that SRH protein could carry out its anti-atherosclerosis through the inhibition of thrombin.4. In order to examine the metabolism of SRH in vivo, polyclonal antibodies to SRH in rabbit and mice were prepared. And we established double antibody-sandwich ELISA to determine the contents of SRH and SAK. The results indicated that half-life of SRH tended to be extended. This laid a foundation for the further exploration of its pharmacy.
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