| Cromer, Knops and Indian blood group systems are the 21st,22nd and 23rd in the classification of International Society of Blood Transfusion (ISBT), respectively. Research about the three blood group systems has been promoted to an even higher level oversea; however, we haven't seen any data about the three blood groups in Chinese people. The capital purpose of our research is to raise the gene detection methods of these three blood groups, and analyze the gene polymorphisms in Chinese people. Additionally, DAF (or CD55) and CR1 (or CD35), which are the blood group proteins of Cromer and Knops, both belong to the family of complement regulatory proteins (CRPs), as well as CD59. Approximate 1% erythrocytes will be cleared in human body every day. There have been many different theories concerning the senescence erythrocytes recognized and cleared by macrophages. By measuring the quantity of the CD55, CD35 and CD59, we aim to know if the CRPs take an important role in the process of senescence erythrocytes clearance.Firstly, the Cromer, Knops and Indian blood groups types were studied with gene sequencing and polymerases chain reaction-sequence-specific primer (PCR-SSP) in Han, Uighur, Tu, Lisu and Dong people of China. Two polymorphism sites locating at introns were found for Cromer and Indian blood groups respectively, while two single nucleotide polymorphism (SNP) sites (A4646G and A4870G) were detected in exon 29 in Knops blood groups and gene frequencies were also calculated. We found that the allele frequencies of these two SNP sites are different in five minority ethnic groups. Other Han samples collected by random were reverse-transcribed to full length cDNA, which were sequenced to discover new gene polymorphism sites in Chinese. As a result,12 foregone SNPs were detected in Chinese Han people.Additionally, with flow cytometry, we detected the surface quantity of CD35, CD55 and CD59, as well as the amount of binding auto-antibody IgG, for the erythrocytes which were obtained by capillary centrifugalization or conserving in vitro for 1 week in 4℃and 37℃. The THP-1 cells were cultured to see if the RBCs can be phageocytized. Comparing with the young RBCs obtained by capillary centrifugalization, the quantity of three kinds CRPs of old RBCs decreased remarkably, and the sensibility of auto-antibody IgG increased. However, for the RBCs conserving in 4℃and 37℃, the number of RBCs surface CD35 and binding auto-antibody IgG didn't change a lot, but the CD55 and CD59 decreased significantly for 37℃conserving RBCs. And a small percentage of old RBCs obtained by capillary centrifugalization could be phagocytized by THP-1 cells, neither did the young RBCs.In a word, we analyzed the distribution of three blood groups in Chinese Han people and several minority ethnic groups, and found the PCR-SSP method to detect Knops blood group type quickly. For the other aspect, most researches referring to senescence RBC phagocytosis focus on auto-antibody and CD47 which regulates the non-opsonization phagocytosis, while little studies are about complement regulatory proteins. Otherwise, we compared the old RBC obtained by capillary centrifugalization and the RBC conserved in vitro from a few aspects which was another novelty.Red blood cell, whose senescence and apoptosis mechanism is different with the "Program Death" of karyocytes, is a kind of special cell in human body without nuclear. Making clear the changes of RBC membrane proteins during the senescence progress, and elucidating the molecule mechanism of senescence and phagocytosis will be very important for the cell apoptosis study. Now, we realize that as RBCs become aging, the CRPs decrease. But whether these descents lead the RBC senescence, and the mechanism of the contribution, more researches are needed to prove. |
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