Study Of Riboflavin Photochemical Inactivate Viru In Red Blood Cells

Pathogen inactivation of blood to enhance blood safety is a way. From the handle onto the object classification, divided into plasma pathogen inactivation, platelet pathogen inactivation and red blood cell pathogen inactivation. Platelet and plasma as no cellular element contained in these blood components, the pathogen inactivation technology is relatively mature, some methods have been applied in clinical. Red blood cells as a cell formed elements more sensitive to of the physical and chemical factors,so the technical development of red blood cells inactivated more slowly. Some scholars use of methylene blue photochemical and psoralen derivatives S-303 inactivated virus in RBC can be effectively kill the model virus.Methylene blue photochemical inactivated model viruses resulted much decrease in RBC ATP and 2,3-DPG level,more 2% hemolysis, besides methylene itself is toxic.Researcher found in animal experiment specific antibody for S-303.Thus these above two methods are not successful. Riboflavin photochemical pathogen inactivation of red blood cells has attracted increasing attention.Riboflavin is known to interact with Nucleic acid and to covalent linkages and breakages on exposure to ultraviolet and or visible light thereby preventing transcription and relication. Since UV light is absorbed by hemoglobin ,preventing the intercalation of the riboflavin into nucleic acid structure.So UV light is inappropriate to activated light source for RBC pathogen inactivation.So this study build a riboflavin photochemical method to inactivate virus in RBC.Objective (1) To research and develop a photochemical methed that can inactivate virus in red blood cell using riboflavin plus visible light.Explore the job of riboflavin concentration and energy of light on effect of inactivation.(2) To investigate model virus nucleic acid degradation after riboflavin photochemical treatment.(3) To study on riboflavin photochemical treatment of red blood cells in vitro the impact of quality indicators.Metheds: (1) Using 400-500nm wavelength of visible light as activatied light source,Human cytomegalovirus(HCMV),as a model virus. Adding HCMV respectively to red blood cell suspensions containing different final concentration of 0,50,150 and 200μmol/L riboflavin, irradiated by a dose of 0,30,60,90, and 120 J/cm2 ( 0,10,20,30,40 min) visible light irradiation. Titer and ctopathic effect was measured ,to evaluated the virus inactivation .Mixture of riboflavin and virus suspensions treated with different riboflavin concentration and illuminating time was collected and inoculated in single layer cells. And the virus titer was measured. The inactivation effects of UV light plus riboflavin for VSV was stuied. Then we choose the best inactivated conditions.(2) The HCMV UL54 genes before and after the conservative region is divided into five overlapping segments, designed five pairs of primers amplified HCMV nucleic acids by the different conditions under treatment.We analysis nucleic acid integrity, observate of riboflavin photochemical rupture of the role of virus nucleic acid,cell culture was performed at the same time as control.(3) Selected conditions for erythrocyte riboflavin photochemical treatment. Quality indicators of red blood cell such as ATP ,2,3-DPG, K + leakage and free hemoglobin can be determinate during the 35 stored days.Results: (1)Riboflavin in a certain range of doses greater the inactivated faster. With a riboflavin concentration of 150μmol/L and visible light intensity of 60J/cm2,the titer of HCMV in red blood cell susepsion could be reduced from 7.65±0.60 lg TCID50 to <0.43lg TCID50.(2) HCMV nucleic acids can not be amplified out gradually. With the inactivation process, and all nucleic acid-PCR results were negative.(3) Best experimental conditions, after treatment, compared with the control group, red blood cells at 35 days storage period, a slight change in the quality indicators.Conclusions: (1)400-500nm wavelength of visible light as activatied light source of riboflavin photochemical method can effectively inactivated virus in red blood cells suspension.(2) Riboflavin photochemical can make a gradual degradation to virus nucleic acid chain.(3) Riboflavin photochemical treatment on erythrocyte function has some influence, but it will not significantly affect the efficacy of infusion.