| So far, over300inherited blood group antigens have been found on the surface of human red blood cells. However, some individuals do not express certain the blood group antigens which are usually expressed by others, such as s, Dib, k antigens. If patients with rare blood phenotypes were repeatedly transfused with unmatched blood, they might produce alloimmune responses, which even would be life-threatening. Therefore, systematic screening of blood groups of donors and recipients is needed to supply compatible blood products. Serological methods are the classical method for testing the blood group antigens and antibodies, but some rare antibodies are not available, or weakly reactive, so it is difficult for serological methods to be used widely in this area. With the continuous development of molecular biology, most of the molecular mechanisms of blood group antigens have been clarified. And most of the blood group antigen polymorphisms are caused by single nucleotide mutation. We put our focus on the gene detection of blood group antigens in this study.The multiplex PCR, several blood group alleles were amplified simultaneously in one system, was used for blood group antigens genotyping. The presence or absence of amplification bands indicates whether the high-frequency antigens were absent or not. Using site-directed mutagenesis (SDM) technique, we successfully prepared the plasmids containing mutational single nucleotide polymorphism(SNP) sites, as negative controls in Dib, k, Jsb blood group antigens genotyping system.Prepared standard plasmids and genomic DNA samples in which some antigens are missing were used to verify the repeatability and stability of the established multiplex PCR system. After the system was well established,4190blood samples from random donors were genotyped for the alleles Dib, k, Jsb1910and Jsb2019. We found that the frequency of Dib, k, Jsb1910and Jsb2019alleles in the cohort study population are all less than0.1%. Two Di(b-) samples were found in our study, and no expression of Dib antigen was detected on the red blood cell membrane of the probands by using serological methods. The homozygous2561C>T mutation was confirmed in both Di(b-) samples by sequencing, hence the frequency of Dia and Dib alleles which produced by the SLC4A12561C>T polymorphism in the cohort study population were0.0219and0.9781respectively. In addition, family analysis was performed for one Di(b-) sample by flow cytometry and sequencing methods. The results of flow cytometry are consistent with that of the sequencing, i.e. the phenotype of the proband is Di (a+b-), the phenotypes of his parents are Di (a+b+), in line with Mendelian genetics.The Ii blood group collection consists of two antigens, i antigen and I antigen, and the epitopes are carbohydrate structure carryied on glycolipids and glycoproteins. The i antigen is a straight chain of N-acetylgalactosamine repeating units, whereas the I antigen is characterized as the branched chain which is branched by the β-1,6-N-acetylglucosaminyltransferase. The conversion of i to I is accompanied by the erythroid differentiation, and parallel with the appearance and disappearance of the transferrin receptor on reticulocyte. So we speculated that the i antigen mainly expressed on normal adult reticulocytes. Consequently, in this study fresh peripheral blood samples of random donors were selected to enrich the younger red cells rich in reticulocytes and the elder red cells at the proximal and the distal end respectively by the capillary centrifuge method. The expression of i antigen was detected on the erythrocytes at the proximal and distal end using serological methods and flow cytometry. The results showed that the intensity of the agglutination reaction for the red cells from the proximal end, the distal end and the whole blood reacting with anti-i antibody is basically the same (the scores of agglutination intensity were about 3). No significant difference could be found in the flow cytometry results. Therefore, it was demonstrated preliminarily that the expression of the i antigen on normal adult red blood cells was irrelevant with the amount of reticulocytes.Then we studied the expression of i antigen on red blood cells of pregnant women by using flow cytometry. And we found that the expression of i antigen on pregnant women’ red blood cells is47.56%over normal adults’red blood cells. And the expression of i antigen decreased as gestational weeks proceeded,28th weeks of pregnancy decreased by20.23%compared with20th weeks of pregnancy,36th weeks of pregnancy decreased by19.60%compared with28th weeks of pregnancy, respectively. Yu and Inaba have reported that three kinds of β-1,6-N-acetylglucosam-inyltransferase(IGnT) transcripts, IGnTA, IGnTB, and IGnTC, could be coded by the human I locus. The formation of I branching on red blood cells is determined by IGnTC. Therefore, the reticulocytes of pregnant women during different gestational period (20,28and36weeks) were purified by CD71MicroBeads, and the RNA samples were extracted and submitted to cRNA synthesis. Subsequently, the expression of IGnTC was measured by semi-quantitative to explore whether the i antigen and I antigen are reciprocal during pregnancy. The results showed that the grayscale ratios of IGnTC/β-actin were1.42,1.68and1.89in the20th,28th and36th week of pregnancy, respectively. The results indicated that in late pregnancy, the expression of IGnTC gradually increased after the20th week of pregnancy. The reverse conversion of I antigen to i antigen might exist during pregnancy. These findings could be helpful for us to further study the signaling pathways of straight-chain i to branched-chain I on red blood cells, and the causes and the pathological mechanisms of blood diseases which have the reverse conversion of I antigen to i antigen. |
PDF Full Text Request