Preliminary Study Of Amifostine In Promoting The Differentiation Of Dami Cells

Background：As a pan-cytoprotective agent, Ethiofos was widely used for organprotection in clinical radiotherapy or cytotoxic chemotherapy. However, amifostinealso had other functions including hematopoietic growth factor effects, inducingtumor cell apoptosis, inhibition of cell cycle progression, etc. which cannot beexplained by classical pathway of alkaline phosphatase and remains to be clarify. Ourclinical studies have found that amifostine based combined regimen achieved a cleareffect in the treatment of chronic immune thrombocytopenia(CITP) andmyelodysplastic syndrome patients, and bone marrow biopsy proved megakaryocytesmaturation disorder improved in patients with CITP, but the mechanism is not clear.We found amifostine may have a role in promoting the differentiation of cells bybioinformatics. This study intends to prove the differentiation of Dami cells tomegakaryocytes and provide laboratory evidence of our clinical studies.Methods：We predict whether amifostine has a role in promoting the differentiation ofcells or not by bioinformatics. The proliferation of Dami cells was determined by cellcounting with different concentrations of amifostine and the optimal concentrationwas screened. The expression of CD33, CD34and CD41a was detected by flowcytometry to identify the differentiation stage of Dami cells. In treatment withamifostine(0.1mmol/L) for0,4,8,12days, Dami cells morphology were observedunder inverted microscope and transmission electron microscope and nuclear wasobserved by Giemsa staining. The expression of CD41a, CD33and DNA ploidy weredetected by flow cytometry at that4point in time. The expression of transcriptionfactor including GATA-1, Fli-1, aNF-E2, fNF-E2at mRNA and protein level weredetected by real time quantitative PCR and Western blot.Results：1) Amifostine may regulate cell differentiation according to bioinformatics,and we find8genes related to promoting cells differentiation;2) Amifostine (>1mmol/L) could inhibit the proliferation of Dami cells and it could promote theproliferation of Dami cells at concentration of0.1mmol/L;3) Amifostine(0.1mmol/L)could promote the proliferation of Dami cells from primitive state towards maturemegakaryocytes. Platelet demarcation membrane system appeared, percent of cells’diameter>20micrometer increased by24.63%, the mean expression of CD41aincreased by206.43, the expression of CD33decreased by13.98%and DNA ploidyincreased during this process.4) As for the expression of transcription factor includingGATA-1, Fli-1, aNF-E2, fNF-E2at mRNA and protein level, no significant increasingwere found.Conclusion：1) Amifostine could promote the differentiation of Dami cells at lowconcentration(0.1mmol/L), and inhibit Dami cells’ proliferation at high concentration(>1mmol/L).2) Dami cells differentiation were proved by the increasing in size,the appearance of platelet demarcation membrane system, the increased expression ofCD41a, and the decreased expression of CD33and increased DNA ploidy.3) Theexpression of transcription factor including GATA-1, Fli-1, aNF-E2, fNF-E2atmRNA and protein level did not increase, and its’ transcriptional activity needs furtherverified.