The aim of study was to explore the potential of human erythroid membrane associated protein (ERMAP) gene in erythroid cell differentiation and development, mononuclear cells (MNCs) were isolated from umbilical cord blood and induced to erythroid cell differentiation by SCF, IL-3 and EPO. The cell morphology was observed by using optical microscopy, the positive rate of cells was counted by biphenylamine staining and the ratios of CD36+/CD235a-, CD36+/CD235a+, CD36-/CD235a+ cells were detected by flow cytometry, the change of human ermap gene expression level was analyzed by using fluorescent quantitative PCR (FQ-PCR). The results showed that the ermap gene expression level increased while MNCs were induced to erythroid lineage after treatment with SCF, IL-3 and EPO. It is concluded that the human ermap gene plays an important role in differentiation and development of erythroid cells.
Blood Group Antigens ; genetics ; metabolism ; Butyrophilins ; Cell Differentiation ; genetics ; Cells, Cultured ; Erythroid Cells ; cytology ; Fetal Blood ; cytology ; Humans ; Leukocytes, Mononuclear ; cytology ; metabolism ; Polymerase Chain Reaction ; methods

To develop a real-time FQ-PCR method for quantifying human ermap, a set of primers and a fluorescent probe were designed by primer express 2.0. pBluescriptSK(+) plasmid contained ermap cDNA was transcribed to generate calibration standards for quantification. A real time FQ-PCR method was established. The results showed that when the concentrations of DNA to be amplified were ranged from 1.725 x 10(7) to 1.725 x 10(10) cps/ml, there was a good correlation between template concentration and cycle threshold, and the correlation coefficient reached to -0.999376. In conclusion, real time FQ-PCR which is specific, sensitive and accurate can be used to further research on human ermap.
Blood Group Antigens ; genetics ; Butyrophilins ; DNA, Complementary ; chemistry ; genetics ; Fluorescent Dyes ; chemistry ; Fluorometry ; methods ; Humans ; Polymerase Chain Reaction ; methods ; Reproducibility of Results

In order to investigate the potential role of human ERMAP gene in erythropoiesis, the ERMAP-dsDNA was designed, ERMAP-shRNA expressing plasmids was constructed, and ERMAP-shRNA/K562 cell was established. Cell morphology, biphenylamine staining, expression of cell surface antigens as well as quantitative level of human ERMAP gene were observed during K562 cells differentiating toward erythroid lineage induced by Ara-C. The results showed that at 72 hours after Ara-C treatment, ERMAP-shRNA/K562 cell size became large with increasing cytoplasm content. The percentage of biphenylamine positive cells increased from 1.17% to 2.04% (p < 0.05), but still lower than that in group K562 + Ara-C. The percentage of CD36(-)/CD235a(+) increased from 8.83% to 11.28%, CD36(+)/CD235a(+) increased from 1.23% to 2.64%, and CD36(+)/CD235a(-) increased from 0.59% to 1.47% respectively, which were all lower than that in group K562 + Ara-C at either time point. At the same time, the level of ERMAP expression increased slowly from 2.52 x 10(-3) to 4.53 x 10(-3), which was also significantly lower than that of group K562 + Ara-C. It is concluded that the ERMAP-shRNA inhibits the Ara-C-induced erythroid differentiation of K562 cells, which further suggests that there is relationship between hERMAP and erythroid differentiation and development.
Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; Cytarabine ; pharmacology ; Erythropoiesis ; drug effects ; Humans ; K562 Cells ; RNA, Messenger ; RNA, Small Interfering ; pharmacology

Human ERMAP (hERMAP) is a novel gene coding for erythroid membrane-associated protein, which may play a role in erythropoiesis. To explore the role of hERMAP in fetal hematopoiesis, 29 fetuses of inevitable abortion with the fetal ages of 9 - 36 weeks were collected, and the total RNAs were isolated from the liver, spleen, kidney, heart, lung, thymus, brain, bone marrow and skeletal muscle, the RNAs from which at 25(+5) week fetal age were used to detect hERMAP expressions in different fetal tissues with Northern blot, and the hERMAP in various fetal tissues were quantified by FQ-PCR. As a result, Northern blot showed that hERMAP was expressed in liver and bone marrow at 25(+5) fetal age, but not in the other organ tissues. FQ-PCR results indicated that the hERMAP had been still expressed in 9 - 36th week fetal liver, increased starting from 12th-week, reaching a peak between 18 - 20th week and declining slowly starting from 21st-week. In the fetal bone marrow, expression of the hERMAP began from 15th week, reached a highest level between 27 - 32nd week and fell rapidly from 33rd week, the expression level of which was lower than that in liver. The low level expression of this gene was observed in some specimens of kidney, heart, lung, thymus, brain and skeletal muscle. It is concluded that the expression of hERMAP in fetal liver approximately consistent with haematopoiesis of fetal liver, and the expression of this gene in bone marrow aged 15 - 32nd fetal weeks coincides with haematopoiesis of fetal bone marrow. It suggests that the function of the hERMAP is possibly related with the migration of erythroid cells to liver and bone marrow during the fetal development process.
Blood Group Antigens ; biosynthesis ; genetics ; Bone Marrow ; metabolism ; Butyrophilins ; Fetus ; metabolism ; Gene Expression ; Gestational Age ; Hematopoiesis ; Humans ; Kidney ; metabolism ; Liver ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Spleen ; metabolism

BACKGROUNDMolecular testing is more precise compared to serology and has been widely used in genotyping blood group antigens. Single nucleotide polymorphisms (SNPs) of blood group antigens can be determined by the polymerase chain reaction with sequence specific priming (PCR-SSP) assay. Commercial high-throughput platforms can be expensive and are not approved in China. The genotype frequencies of Kidd, Kell, Duffy, Scianna, and RhCE blood group antigens in Jiangsu province were unknown. The aim of this study is sought to detect the genotype frequencies of Kidd, Kell, Duffy, Scianna, and RhCE antigens in Jiangsu Chinese Han using molecular methods with laboratory developed tests.

METHODSDNA was extracted from EDTA-anticoagulated blood samples of 146 voluntary blood donors collected randomly within one month. Standard serologic assay for red blood cell antigens were also performed except the Scianna blood group antigens. PCR-SSP was designed to work under one PCR program to identify the following SNPs: JK1/JK2, KEL1/KEL2, FYA/FYB, SC1/SC2, C/c and E/e.

RESULTSSerologic antigen results were identical to the phenotypes that were predicted from genotyping results. The allele frequencies for Jk*01 and Jk*02 were 0.51 and 0.49, respectively; for Fy*A and Fy*B 0.94 and 0.06; for RHCE*C and RHCE*c 0.68 and 0.32; and for RHCE*E and RHCE*e 0.28 and 0.72. Among 146 blood donors, all were KEL*02/KEL*02 and SC*01/SC*01, indicating allele frequencies for KEL*02 and SC*01 close to 1.00.

CONCLUSIONSThe use of PCR-SSP working under the same condition for testing multiple antigens at the same time is practical. This approach can be effective and cost-efficient for small-scale laboratories and in developing counties. These molecular tests can be also used for identifying rare blood types.

Blood Group Antigens ; genetics ; Butyrophilins ; China ; ethnology ; Duffy Blood-Group System ; genetics ; Gene Frequency ; Genotype ; Humans ; Kell Blood-Group System ; genetics ; Kidd Blood-Group System ; genetics ; Polymerase Chain Reaction ; Polymorphism, Single Nucleotide ; Rh-Hr Blood-Group System ; genetics

To investigate the pattern of human ERMAP gene expression in different cell lines, 15 cell lines derived from hematopoietic tumor, somatic tumor and normal tissue were chosen and were cultured; cells were harvested after culture for 12, 24, 36, 48, 60 and 72 hours; the expression of the human ERMAP was detected by using fluorescent quantitative PCR. The results showed that human ERMAP gene expression was positive in K562 cell line at interval of 12 and 24 hours, and the expression levels were (5.092 +/- 2.331) x 10(6) cps/microl RNA, (5.328 +/- 3.916) x 10(6) cps/microl RNA, respectively; ERMAP gene expression was also positive in ECV304 cell line at interval of 24 hours, and the expression level was (0.84 +/- 0.12) x 10(6) cps/microl RNA; and its expression was negative in other 13 cell lines. It is concluded that human ERMAP gene expression in ECV304 cell line was found first, and its expression in K562 cell line was further confirmed.
Blood Group Antigens ; genetics ; Butyrophilins ; Cell Line ; Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; HL-60 Cells ; Hematologic Neoplasms ; genetics ; pathology ; Humans ; Jurkat Cells ; K562 Cells ; RNA, Messenger ; genetics ; metabolism

In order to investigate the potential of human ERMAP gene in erythroid cell differentiation, K562 cells were induced to erythroid lineage by Ara-C and to macrophage lineage by TPA, human ERMAP mRNA was detected by fluorescent quantitative PCR. The results showed that human ERMAP mRNA increased while K562 cells were induced to erythroid lineage after treatment with Ara-C at 2.5 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. Human ERMAP mRNA not changed while K562 cells were induced to macrophage lineage after treatment with TPA at 2.0 x 10(-6) mmol/L/L and 1.0 x 10(-6) mmol/L/L. It is concluded that human ERMAP gene plays an important role in differentiation and proliferation of erythroid cells.
Antigens, CD ; analysis ; Antigens, Differentiation, Myelomonocytic ; analysis ; Blood Group Antigens ; genetics ; Butyrophilins ; Cell Differentiation ; drug effects ; genetics ; Cytarabine ; pharmacology ; Erythrocytes ; cytology ; metabolism ; ultrastructure ; Flow Cytometry ; Gene Expression ; drug effects ; Humans ; K562 Cells ; Macrophages ; cytology ; metabolism ; ultrastructure ; Microscopy, Electron ; RNA, Messenger ; biosynthesis ; genetics ; Receptors, Transferrin ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Time Factors