The Function Of P38-2G4in The Erythriod Differentiation Of Mice

Erythroid differentiation is a process of complex and consecutive changes. Normally, the mammalian hematopoietic stem cells in the bone marrow have several phases, including pro-erythroblast, basophilic erythroblast, polychromatic erythroblast, orthochromatic erythroblast, reticulocyte and erythrocyte that can cross the blood brain barrier to reach the blood. The terminal differentiation was mainly composed by five characterizations; cell division-stopped, cell volume-decreased, nuclear pyknosis, denucleation and expression of globin gene. With the action of several carcinogens, the erythroblasts loose the ability to differentiate to the mature erythrocyte but stay in the certain stage that caused the diseases or leukemia.Making a deep studying to the process of erythroid differentiation has been a primary focus of many researchers’work. Our group emphasized on the study of erythroid differentiation and the pathogenesis of leukemia. As in the earlier work, the total extract of fetal liver cells, FVA cells and MEL cells induced by1.2%DMSO were analyzed by two-dimensional electrophoresis technology, combined with mass spectrometry technology to identify the erythroid differentiation-related proteins. We have found a special protein named p38-2G4, which showed significant changes in protein expression.p38-2G4was first recognized as a DNA binding protein in Ehrlish ascites tumor cells. However, there is no research about the function of p38-2G4at present, while concentrated in the functional research of its human homologue (Ebpl). Ebp1has a wide effect on cell function including proliferation, differentiation, apoptosis and etc. It has a high similarity in the sequence of amino acid with p38-2G4. They both have394amino acids, and different in four amino acids.This paper plans to do some functional research on p38-2G4during the murine erythroid differentiation to study the effect of this protein family on erythroid differentiation. Western blot to confirm the expression of p38-2G4in fetal liver, FVA and MEL cells induced by1.2%DMSO. On this basis we are supposed to study the function of p38-2G4. Firstly, Western blot to detect the expression in several murine organs and erythriod cells. Immunocytochemistry to identify the subcellular location of p38-2G4protein. Thirdly, studying the function of p38-2G4in model of MEL induced by sodium butyrate (SB). Western blot and RT-PCR to detect the expression of p38-2G4during the differentiation of MEL induced by SB. Moreover, to further study the effect of p38-2G4on proliferation, differentiation of MEL, lentiviruses were used to change the expression of p38-2G4in MEL cells. For this, two pairs of shRNA were determined according to the coding region of p38-2G4mRNA. Using the method of Molecular cloning to establish the recombinant lentivirus vector shRNA1/shRNA2-pLKO.1-TRC and p38-2G4-pLJM1. They were transfected to HEK293T cells coupled with packaging plasmid (pCMV-VSV-G, pCMV-dR8.2) separately. Incubated with48h, the supernatant was collected and added to the MEL cells. After puromycin selection, cells with steadily low/high expression of p38-2G4were applied to the study of erythroid differentiation. MTT assay was used to identify the effect of p38-2G4on cell proliferation. Flow cytometer was used to identify the effect of p38-2G4on cell cycle. Furthermore, SB inducing and Benzidine staining were used to identify the effect of p38-2G4on erythroid differentiation.Western blot shows that p38-2G4has two isoforms, and expressed widely in murine organism and erythroid cells. However, the distribution of isofome is different in organism and cells. Both Western blot and RT-PCR show that p38-2G4has changed its expression during erythroid differentiation with the tendence of down-regulated. In addition, the result of western blot reveals the stable cell strain is eatablished successfully. MTT assay show that p38-2G4cannot affect the cell proliferation. However, Benzidine staining shows that both high expressed and low expressed on the protein level of p38-2G4can affect the synthesis of hemoglobin.In a conclusion, p38-2G4has an important role in erythroid differentiation. However, it has no significant influence on MEL cells proliferation. Therefore, this project aims to do a further study of mechanism of p38-2G4participates in erythroid differentiation. We have established the recombinant Tandem Affinity Purification vector (p38-2G4-V51-S3). Aiming to use TAP experiment and Mass spectrometry to identify the p38-2G4-interacting proteins during erythroid differentiation.