Experimental Study Of Exoglycosidases Clearing Blood Group Antigens In Human Organs

Objective:Experimental study exoglycosidases to remove human blood group antigens of human red blood cells and liver and explore the feasibility of converting the blood type in human organ;Methods:一、The a-galactosidase, resource of green coffee beans, was used to clear human blood type B antigens on the surface of human erythrocyte;The optimum reaction temperature of a-galactosidase is25℃, the optimum pH is6.5and the best reaction solution is potassium phosphate buffer solution. Different dilutions of a-galactosidase have different effect in removing blood type B antigens on red blood cells, the experiment is to screen the optimal enzyme reaction concentration.10-fold diluted a-galactosidase, which was diluted by potassium phosphate solution, was used to treatment blood type B erythrocytes60mins. Using flow cytometry to analysis the effective of a-galactosidase clearing B antigens on the red blood cell; To compare the changes of the enzyme activity, at different condition of temperatures (4℃,25℃);The patient livers resected in liver transplantation were used as experimental subjects and established the liver perfusion model. With UW solution+/-α-galactosidase in vitro perfusion type B liver specimens, and preserved4hours, immunofluorescence analysis of changes in liver tissue blood group antigens and explore a-galactosidase scavenging the blood group antigens of human livers from organ level; Experimental groups and control groups, use ANOVA to analyse levels of blood group antigens in different time periods within the group, and compare blood group antigen levels are statistically significant differences with group. The same time the level of blood group antigens between group independent samples t-test, and compare between blood group antigen levels the same point in time whether the difference was statistically significant.二、The recombinant a-N-acetyl-galactosidase to clear A antigens on the surface of human erythrocyte;5%blood type A erythrocytes suspension was prepared, which were treatment by a-N-acetyl galactosamine60min. Flow cytometry analysis, drawing blood group antigen clearance curve with different enzyme concentration, and screen the optimal enzyme clears blood group antigen concentration of enzyme reactions. To compare the changes of the enzyme activity, at different condition of temperatures (4℃,37℃);The patient livers resected in liver transplantation were used as experimental subjects. With UW solution+/-α-N-acetyl-galactosidase in vitro perfusion type A liver specimens, and preserved4hours, immunofluorescence analysis of changes in liver tissue blood group antigens and explore a-galactosidase scavenging the blood group antigens of human livers from organ level; Experimental groups and control groups, use ANOVA to analyse levels of blood group antigens in different time periods within the group, and compare blood group antigen levels are statistically significant differences with group. The same time the level of blood group antigens between group independent samples t-test, and compare between blood group antigen levels the same point in time whether the difference was statistically significant.Results:(1) Different dilutions of the a-Gal have different efficiency on removal blood group antigens of RBC;10-fold dilution of the a-Gal was the best optimal enzyme reaction concentration on remove B antigens; Low temperature reduced a-galactosidase efficiently removing blood group antigensWith UW solution+α-Gal in vitro perfusion blood type B liver specimens, preserved4hours, B antigens on liver specimens after perfusion significantly reduced. Experimental groups use ANOVA to analyse levels of blood group antigens in different time periods within the group, P values<0.01, the difference were statistically significant. Experimental group B antigen average level in liver tissue1h decreased to58%,2h was10%,4h was4%.(2) Different dilutions of the α-NAGA have different efficiency on removal blood group antigens of RBC;100-fold dilution of the α-NAGA was the best optimal enzyme reaction concentration on remove B antigens; Low temperature reduced a-galactosidase efficiently removing blood group antigensWith UW solution+a-Gal in vitro perfusion blood type B liver specimens, preserved4hours, Aantigens on liver specimens after perfusion significantly reduced. Experimental groups use ANOVA to analyse levels of blood group antigens in different time periods within the group, P values<0.01, the difference were statistically significant. Experimental group B antigen average level in liver tissue lh decreased to46%,2h was15%,4h was3%.Conclusions:(1) a-galactosidase can effectively remove blood group B antigens on the surface of red blood cells and achieve blood type conversion from B�'O; UW solution+α-galactosidase in vitro perfused type B liver specimens, and the livers were preserved4hours, B antigens in liver tissues significantly reduced, but did not reach undetectable levels;(2) a-N-acetyl-galactosidase can effectively remove blood type A antigen on the surface of erythrocyte and achieve blood type conversion from A�'O; UW solution+α-N-acetyl-galactosidase were in vitro perfused blood type A liver specimens, and the livers were preserved4hours, blood group A antigens of liver tissue significantly reduced.