Study On Preparation, Quality Standards And Application Of CVF

Aim:Objective to lyophilized freeze-dried formulations of CVF products,the establishment of quality standard of complement proteins CVF,as well as related research which used for establish endothelial inflammation injury model related to the expression of fibrinolysis and coagulation.Methods : Cobra venom factor was separated and purified by ion exchange chromatography, molecular sieve, anion exchange chromatography, and its purity was identified by isoelectric poly, alkaline gel electrophoresis, and high performance liquid chromatography. The effect of different protein concentrations(0.200,0.500,1.000mg/m L),protective agents(sucrose,lactose,trehalose,mannitol,BSA),p H value(6.0,6.8,7.4) on freeze-drying process of CVF were investigated, and the effect of different temperature( 6,25,37°C) on stability of freeze-drying CVF was also investigated. We programmed the preparation and established the quality standards of CVF based on the above research. CVF was used to activated complement alternative pathway of serum specifically,after exposure of HMEC to activated complement for various times,supernatant was removed and assayed for expression of P-selectin,VWF,t-PA,PAI-1,TF,TM,and NO by using reagent kits. The expression of the above molecular in HMEC pretreated with PDTC and resveratrol were also investigated. Results : Purified CVF showed a single band in alkaline gel electrophoresis and isoelectric polyethylene. CVF with higher concentrations showed higher activity and protein quantity than that of CVF with lower concentrations during freeze drying. Single protective agent sucrose,lactose, trehalose,mannitol had aprotective effect on protein quantity and activity of CVF,BSA protected protein quantity and activity of CVF,and the difference was statistically significant(P <0.05 or P <0.01) compared with normal group. Mannitol combined with trehalose,sucrose and lactose, respectively protected protein quantity and activity of CVF(P <0.05),and combination group showed a better protective effect on CVF with higher concentration,and no significance was showed among the three formulations. Lower p H value showed the effect of reduce protein quantity and activity of CVF during freeze-drying process of CVF. In the study of stability, trehalose-mannitol combination group showed a better effect on stability of freeze-drying CVF.P-selectin and VWF were rapidly released by endothelial cells and the expression reached the peak at the time point of 15 min, the expression of t-PA, PAI-1,and TF were continuously upregulated. Whereas NO and TM were decreased. Moreover,PDTC and resveratrol inhibited the upregulation of P-selectin,VWF, t-PA, PAI-1, and TF, intervened the downregulation of NO,but resveratrol further downregulated the expression of TM. Conclusion : The frozen dried technology of CVF which established in this study was viable, stable, and controllable, and quality standar was established, it could provide guidance and reference for research of CVF in the future. In human microvascular endothelial cells inflammation induced by the activation of complement alternative pathway,the change of molecule expression related to coagulation and fibrinolysis and the intervention of PDTC and Resveratrol,it could be provide scientifical evidence and reference.