The Effect Of GSK3 And P38 Signaling Pathway Inhibitors On HSC Amplification And The Observation Of Four Fusion Gene Leukemia Cell Phenotypes

Hematopoietic stem cells(HSCs)are a small group of cells characterized by their ability to self-renew and differentiate into all mature blood cell lineages.They are responsible for maintaining the homeostasis of hematopoiesis system and the function of immune system as well.HSCs transplant is regarded as curative therapy available for patients with high-risk leukemia or other hematologic cancers.However,bone marrow and peripheral HSCs transplantation are limited by difficulty of matching marrow donor or the relatively high risk of transplant-related complications,particularly graft rejection.Umbilical cord blood cells have great advantage in clinical treatment.However,the small quantity of blood obtained from an umbilical cord is hardly enough to meet the demand of hematopoietic reconstitution.So HSCs expansion studies providing new possibilities for the application of HSC transplantation.Previous researches have shown that p38 MAPK and GSK3 inhibition can stimulate hematopoietic stem cell proliferation.Most of these studies focused on sorted LSK cells culture,which is not very convenient for clinical applications.Furthermore,after a series of experiment treatments,cells were damaged at different degree.The purpose of our study is to keep cells in good condition while ensuring expansion efficiency of HSCs.Here,we developed a method to avoid cell damages and simplify procedures by culture whole bone marrow.Glycogen synthase kinase 3(GSK-3)is a serine/threonine protein kinase,recently regarded as key regulator of many signaling pathways.It mainly includes two subtypes: GSK-3? and GSK3?,they are participated in different pathway.Inhibit GSK-3 will promote HSCs proliferation,so we assumed that efficiency of HSCs expansion will be promoted by using specific inhibitor.Firstly,we compared two inhibitors of GSK3 signal pathway and found that inhibitor with higher specificity can actually amplify hematopoietic stem cells more efficiently by flow analysis and CFU.Then,based on the original research in our laboratory,GSK3? signal pathway inhibitor was used in combination with p38 MAPK signaling pathway inhibitor to treat cells.And the optimal concentration of these inhibitors was measured respectively or simultaneously by flow analysis.Finally,competitive transplantations and CFU were performed to further verify whether the multi-directional reconstruction capability ofHSCs expansion was improved.The results showed that GSK3? inhibitors didn't promote the effective expansion of HSCs.It may be that GSK3? and GSK3? is complementary in regulating HSCs.Leukemia is a type of malignant clonal disease of hematopoietic system.leukemia-related gene mutations occurred in hematopoietic stem and progenitor cells are the common reason leukemogenesis,and MLL gene rearrangement is a common gene mutation.The mixed lineage leukemia gene is located on chromosome11q23,and it can be disrupted by chromosomal translocations and other rearrangements.Chromosomal translocations result in the fusion of the amino-terminal part of MLL to one of more than 70 different partners forming gain-of-function oncoproteins.Now,it has been reported that the newly generated fusion gene is closely related to the leukemogenesis.In this experiment we focused on the two MLL-AF9 and MLL-ENL fusion genes.Firstly,we used viral infections to construct four leukemic cell models: MLL-AF9 Long,MLL-AF9 Short,MLL-ENL Long,and MLL-ENL Short,then we used flow cytometry to detect the cell cycle,drug resistance of MTT assay;at the same time,we compared them with colony formation ability,and growth rate.The results showed that the MLL-ENL Long fusion gene leukemia cells were significantly different from those of the other three cells.The reason of this difference in the pathogenesis of leukemia remains to be further studied.