Erythroid Differentiation And Development Of The Process Of Globin Gene Expression Analysis Of The Switching Process And Related Regulatory Factor Genes To Explore

During human development, two switches of globin gene expression, ε (embryonic) →γ (fetal) →β (adult) occur. Adult erythropoiesis is also postulated to recapitulate γ- to β- globin switching, which is called "compressed switch". Although advance has been made in the study on control of globin gene expression and switching, key factors of globin gene switching is still unknown till now. As the validation of these factors is of significance for disclosing mechanisms of globin gene switching, we hope that our work could contribute to this target.In this study, Mono-nuclear cells (MNCs) containing hematopoietic stem/progenitor cells were isolated from umbilical cord blood of normal full-term deliveries, healthy adult and heterocellular HPFH patient bone marrow by density gradient centrifugation. After simple purification and expansion in vitro, MNCs were induced to erythroid cell differentiation using different culture systems and cytokines including Epo, IL-3 and GM-CSF. In the previous study, fetal bovine serum and the other components such as c-kit ligand and Tpo were often used when erythroid cells were cultured in vitro. However, the fetal (γ-) globin gene was reactivated in cultured erythroid cells from adult bone marrow or peripheral blood in these culture conditions. That is, these culture methods failed to mimic the erythropoiesis in physical condition and couldn't meet the needs for study of globin gene regulation and switching. In this study, serum free medium were used for induction of erythroid cells from adult bone marrow and human cord serum-containing medium were used for erythroid cells from cord blood. Further, components that reactivated γ-globin gene were never used in our culture system. We hope that the culture condition and methods mimicking erythropoiesis in vivo could be established and thus, can provide us the materials for further study of globin gene regulation and switching.During induction, erythroid cells were collected from cultures at different time points and total RNA was extracted for reverse transcription reaction. Then the expression level of α-like and β-like globin genes in erythroid cells at different time points were determined by real time PCR. The results showed that the expression level and fluctuation of -α,β- and γ-globin gene in cultured erythroid cells of adult bone marrow is very similar to the so called "compressing switch" in vivo when cultured in serum free medium. Compared to y-globin gene, the expression of β-globin gene is predominant, whereas the expression of 8- (embryonic) and ζ,-(embryonic) globin genes is nondetectable. However, the expression of α-,β-,γ-,...