In-situ Immunofluorescence Study Of Artificial Red Blood Cell Membrane Protein Reconstruction In Vitro And Its Functional Analysis

There are some practical issues to traditional method to solve all the clinical transfusion medicine and battlefield first aid cases, so study of blood substitutes emerge. Development so far, it has been developed three generations of blood substitutes. The third generation of artificial red blood cell is more close to natural red blood cell in its structure and properties, and has ability of carrying and releasing oxygen. This subject is mainly about protein structure, mechanical properties and oxygen-carrying capacity of artificial red blood cell which is reconstructed based on cytoskeleton. What's more, we do some research about locating membrane protein of the process of reconstructing red blood cell according to immunological methods. So that it provides a visual evidence of reconstructing red blood cell in vitro from the microscopic point of view. As well as providing a new way to research on the structure of artificial red blood cell. Objective:Researching on the function and properties of artificial red blood cell, and establishing a methodology about researching the membrane protein structure of erythrocyte which is reconstructed in vitro by immunological methods. Then provide a new information for researching on blood substitutes.Methods : Preparing artificial red blood cells and observing its oxygen saturation-dissociation curve (P50) and viscoelasticity and so on. Analyzing the oxygen-carrying function also physical and chemical properties of artificial red blood cell. By means of immunofluorescence, locating membrane protein of reconstructed artificial red blood cell, so that we can obtain structure and distribution of membrane protein. Analyzing the process of the reconstructing red blood cell by fluorescence localization method, the results further reveal the process.Results:The elasticity and surface-to-volume ratio of artificial red blood cell are both similar to natural red blood cell, its value in the range of natural red blood cell. According to determine oxygen affinity, we got results that P50 of natural red blood cell is 26.93 meanwhile artificial red blood cell is 15.67, there is some different in the numerical value. We can see the oxygen affinity of artificial red blood cells is better than natural red blood cells. By applied this method, we analyzed the artificial RBC reconstructed by our laboratory from the view of membrane structural protein. The conformation of artificial RBC and its reconstrucing processes were analyzed by the comparison of distribution of spectrin in both natural RBC and artificial RBC, while spectrinαl was coloured in situ by indirect immunofluorescence method using fluorescence labeled rabbit-anti-human immunoglobulina and the fluorescence detected with laser scanning confocal microscope (LSCM) was excited by blue light of 488nm wavelength. It is similar to natural RBC, spectrin also distributed in the cytomembrane of RBC. Besides, our results further revealed the reconstructing processes of artificial RBC in vitro.Conclusion:Reconstructed artificial red blood cell is similar to natural red blood cell in its shape. The properties of morphostructure are closely related to its bio-mechanics and function of oxygen carrying-oxygen releasing. The results of cell stability test show that artificial red blood cells can keep stable structure and maintain good physiological and biochemical index. By specific fluorescence localization of membrane protein, it is similar to natural RBC, specific structural protein also distributed in the cytomembrane of RBC which suggest that during the process of cell restructing, the membrane protein arrayed randomly on the basis of biomechanics theory.