Screening and Identifying Reference Genes for Erythrocyte Production from Cord Blood CD34+ Cells Exposed to Hypoxia.

Cord blood (CB) CD34+ cells have the potential to be used to achieve artificial hematopoiesis because of their ability to expand and differentiate in multiple directions. However, the mechanism and molecular changes underlying such differentiation are still unclear. The differentiation of CB CD34+ cells is generally driven by subtle changes in gene expression. A crucial method for examining gene expression is quantitative real-time polymerase chain reaction, but the accuracy of the results is dependent on the use of reliable reference genes. Here, the transcription levels of 10 novel candidate reference genes (EIF4G2, DYNC1H1, LUC7L3, CD46, POLR1D, WSB1, GAPVD1, HGS, LGALS8, and RBM5) and 8 traditional reference genes (GAPDH, YWHAZ, ACTB, B2MG, TBP, HMBS, PPIA, HPRT1) in CB CD34+ cells under different oxygen concentrations were screened and evaluated by using the geNorm and NormFinder algorithms. Comprehensive analysis conducted by RefFinder online tool showed that TBP (a traditional reference gene) and EIF4G2 (a novel reference gene) had the most stable expression, whereas GAPDH and HMBS were the least suitable reference genes under these conditions. These results may serve as a basis for selecting reference genes with stable expression for more accurate normalization under different oxygen concentration stimulation during CB CD34+ cells differentiation.

Gene Expression Profile Reveals Hematopoietic-Related Molecule Changes in Response to Hypoxic Exposure.

The Qing-Tibet Plateau is characterized by low oxygen pressure, which is an important biomedical and ecological stressor. However, the variation in gene expression during periods of stay on the plateau has not been well studied. We recruited eight volunteers to stay on the plateau for 3, 7, and 30 days. Human Clariom D arrays were used to measure transcriptome changes in the mRNA expression profiles in these volunteers' blood. Analysis of variance (ANOVA) indicated that 699 genes were significantly differentially expressed in response to entering the plateau during hypoxic exposure. The genes with changes in transcript abundance were involved in the terms phosphoprotein, acetylation, protein binding, and protein transport. Furthermore, numerous genes involved in hematopoietic functions, including erythropoiesis and immunoregulation, were differentially expressed in response to hypoxia. This phenomenon may be one of reasons why the majority of people entering the plateau do not have excessive erythrocyte proliferation and are susceptible to infection.

Genetic changes in the EPAS1 gene between Tibetan and Han ethnic groups and adaptation to the plateau hypoxic environment.

In the Chinese Han population, prolonged exposure to hypoxic conditions can promote compensatory erythropoiesis which improves hypoxemia. However, Tibetans have developed unique phenotypes, such as downregulation of the hypoxia-inducible factor pathway through EPAS1 gene mutation, thus the mechanism of adaption of the Han population should be further studied. The results indicated that, under plateau hypoxic conditions, the plains population was able to acclimate rapidly to hypoxia through increasing EPAS1 mRNA expression and changing the hemoglobin conformation. Furthermore, the mutant genotype frequencies of the rs13419896, rs1868092 and rs4953354 loci in the EPAS1 gene were significantly higher in the Tibetan population than in the plains population. The EPAS1 gene expression level was lowest in the Han population carrying the A-A homozygous mutant of the rs13419896 locus but that it increased rapidly after these individuals entered the plateau. At this time, the hemoglobin content was lower in the homozygous mutant Han group than in the wild-type and heterozygous mutant populations, and the viscosity of blood was reduced in populations carrying the A-A haplotypes in rs13419896 and rs1868092 Among Tibetans, the group carrying homozygous mutations of the three SNPs also had lower hemoglobin concentrations than the wild-type. The Raman spectroscopy results showed that exposure of the Tibetan and Han population to hypoxic conditions changed the spatial conformation of hemoglobin and its binding ability to oxygen. The Tibetan population has mainly adapted to the plateau through genetic mutations, whereas some individuals adapt through changes in hemoglobin structure and function.

Investigation of the differences between the Tibetan and Han populations in the hemoglobin-oxygen affinity of red blood cells and in the adaptation to high-altitude environments.

OBJECTIVE: High altitude is characterized by low oxygen pressure, resulting in multiple adaptive responses. Tibetans who have lived in the plateau for thousands of years have developed unique phenotypes, such as downregulation of the HIF pathway through EPAS1 and EGLN1 gene mutation. However, the changes of hemoglobin-oxygen affinity under hypoxia environment remain elusive. METHODS: A blood cell analyzer and a blood oxygen analyzer were used to conduct routine blood tests and measure the oxygen affinity P50 in in the Han population that rapidly entered the plateau (for 3-7 days), the plateau-acclimatized Han population (residing for 30 days on the plateau), the plateau Han population (more than 10 years on the plateau), and the Tibetan population. RESULTS: The Han population that rapidly entered the plateau had increasing higher P50 values, RBCs counts and hemoglobin (HGB) levels, while the acclimatized Han population, the plateau Han population and Tibetan all had significantly lower P50 values. However, there were no significant differences in the RBCs counts and HGB levels between the plateau Han, Tibetan populations and the Han population of the plains. DISCUSSION: The adaptability of the Tibetan and plateau Han populations to the plateau was mainly due to the strong affinity of HGB for oxygen, which provided sufficient oxygen for tissues and organs. CONCLUSIONS: The change of P50 could be a feature of the adaptation to the plateau and to avoid altitude sickness, such as high-altitude polycythemia and dyspnea.

Identification of reference genes in blood before and after entering the plateau for SYBR green RT-qPCR studies.

BACKGROUND: Tibetans have lived at high altitudes for thousands of years, and they have unique physiological traits that enable them to tolerate this hypoxic environment. However, the genetic basis of these traits is still unknown. As a sensitive and highly efficient technique, RT-qPCR is widely used in gene expression analyses to provide insight into the molecular mechanisms underlying environmental changes. However, the quantitative analysis of gene expression in blood is limited by a shortage of stable reference genes for the normalization of mRNA levels. Thus, systematic approaches were used to identify potential reference genes. RESULTS: The expression levels of eight candidate human reference genes (GAPDH, ACTB, 18S RNA, ß2-MG, PPIA, RPL13A, TBP and SDHA) were assessed in blood from hypoxic environments. The expression stability of these selected reference genes was evaluated using the geNorm, NormFinder and BestKeeper programs. Interestingly, RPL13A was identified as the ideal reference gene for normalizing target gene expression in human blood before and after exposure to high-altitude conditions. CONCLUSION: These results indicate that different reference genes should be selected for the normalization of gene expression in blood from different environmental settings.

[Analysis for Discordance of Positive and Negative Blood Typing by Gel Card].

OBJECTIVE: To explore the method of Gel card identifying ABO blood group, determine the inconsistent cause and the distribution of disease affecting factors, and put forward a method of its solutions. METHODS: To collect 240 positive and negative typing-discordant blood speciments from patients examined by Gel card and send these speciments to blood type reference laboratory for examining with the classic tube method and serological test, such as salivary blood-group substance, in order to performe genotyping method when serologic test can not be determined. RESULTS: Among 240 positive and negative typing-discordant blood speciments from patients examined by Gel card, 107 blood speciments were positive and negative consistent examined by false agglutination test (44.58%), 133 blood specinents were discordent examined by false agglutination (55.42%), out of them, 35 cases (14.58%) with inconsistent cold agglutination test, 22 cases (9.17%) with weakened AB antigenicity, 16 cases (6.67%) with ABO subtyping, 12 cases (5.00%) with positive direct antiglobulin test, 11 cases (4.58%) with reduced or without antibodies, 11 cases (4.58%) with false aggregation caused by drugs or protein, 11 cases (4.58%) with salivary blood-type substances, 8 cases (3.33%) with non-ABO alloantibody, and 7 cases (2.92%) with allogeneic bone marrow transplantation. The distribution of disease were following: blood disease (16.83%), tumor (11.88%), and cardiopulmonary diseases (11.39%); chi-square test results indicated that the distribution significantly different. CONCLUSION: The analysis of ABO blood grouping shows a variety factors influencing positive and negative blood typing, and the Gel Card identification can produc more false positive blood types. Therefore, more attention should be paid on the high incidence diseases, such as blood disease, tumor, and cardiopulmonary disease.