Based On Low-immunogenicity Ghosts Reconstitute Red Blood Cell Substitutes In Vitro

The role of blood in the clinical first aid is very important,however,the blood resources are in short supply because of its less source and the brief preservation time in vitro,et al,which increases the burden of clinical blood use;at the same time,the emergency blood transfusion is difficult,in the process of needding blood to first aid,it takes time to perform blood type testing and cross-matching in order to avoid blood type agglutination caused by blood type mismatch that is the direct cause of death in patients with blood transfusion.People have been committed to the development of blood substitutes to alleviate the pressure of clinical blood.Research on blood substitutes can be divided into plasma substitutes,platelet substitutes and red blood cell substitutes.Among them,red blood cell substitutes are a research hotspot due to the functions of transporting oxygen,maintaining blood osmotic pressure and expanding blood volume.Objective This subject is a new attempt to prepare ghosts and to convert blood groups.Meanwhile,the prepared ghosts,which are carriers,and the hemoglobin solution are used to reconstitute the red blood cell in vitro to as a potential red blood cell substitutes.Methods The membrane protein extract-rupture buffer for western blotting was used as a hypotonic solution to prepare ghosts.Based on the principle of hypotonicity,red blood cell absorbed water in hypotonic solution which caused red blood cell to swell and appeared aperture.And then,by washing red blood cell continuously in a high-speed rotating to remove intracellular substances of red blood cell and to reduce blood groups antigens on the cell surface,moreover,we prepared ghosts because of the ability of the cell membrane can spontaneously reseal the pore size on the membrane.The western blotting and the blood serum agglutination test were used to detect the ABH antigens on the surface of ghosts;then,the prepared low immunogenic ghosts and hemoglobin solution were used to reconstitute the red blood cell in vitro;finally,we analysed the surface charge,deformation ability and carry/release oxygen function of the reconstituted red blood cell and discussed the possibility of the reconstituted red blood cell to be a potential red blood cell substitute.Results The structure of the ghosts prepared in this study was intact and the hemoglobin clearance rate of ghosts was up to 99.9%.The results of western blotting and serum agglutination test showed that the immunogenicity of ABH antigens on the membrane surface of ghosts prepared by using hypotonic haemolysing solution was weakened.And then,the low immunogenic ghosts and hemoglobin were reconstituted into red blood cell in vitro.The reconstituted red blood cell was intact structure and the surface charge was slightly reduced but the affect was not obvious;by detecting the deformability we found that the deformability of the reconstructed red blood cell was deteriorated and the membrane elongation index（EI）at a shear force of 30 Pa was0.154±0.057（EI of normal red blood cell is 0.6±0.008）,but the hemorheology test showed that the blood substitute containing reconstituted red blood cell had non-Newtonian fluid characteristics,and its blood viscosity-shear rate curve was similar to the blood viscosity-shear force curve of normal whole blood;the results of carrying/releasing oxygen function test showed that the P₅₀（oxygen affinity）of reconstructed red blood cell was to 18 mmHg,indicating that the reconstructed red blood cell had a strong ability to bind oxygen.Conclusion Based on the principle of hypotonic method,this study had prepared the low immunogenic ghosts with intact structure,and had reconstructed red blood cell in vitro by using the low immunogenic ghosts as carriers;although the deformability of reconstituted red blood cell was partially lost,the blood viscosity-shear curve of blood substitute containing reconstituted red blood cell was similar to the normal whole blood’s,which is a prerequisite for exerting its physiological effects.And the affinity of reconstructed red blood cell to oxygen was increased,which could reduce the risk of ischemia-reperfusion injury in patients with emergencies such as hemorrhagic shock,increasing the survival rate of patients and getting more time for continued treatment.In summary,reconstructed red blood cell that we made had some potential as a red blood cell substitute,but it still needs more effort.