Isolation And Culture Leukemia Stem Cells And Its Immune Related Molecular Expression Research

Objective:To isolation and culture leukemia stem cells which are derived from acutemyeloid leukemia patient’s bone marrow and to do the same with hematopoietic stemcells from leukemia-free person’s bone marrow as control. Then to measure theexpression of TGF-β mRNAå'ŒIL-10mRNA in the above two stem cell samples ofpopulations and invest the difference between them. It may give help to study theacute leukemia stem cells’ role of immune-escape mechanism in acute leukemia.Methods:The mononuclear cells of acute myeloid leukemia and leukemia-freeperson’s bone marrow were collected with Lymphocyte separation liquid, and werewashed with PBS buffer and then suspension cultured or directly detected by flowcytemetry after stained with fluorescent antibody. Then the leukemia stem cells aswell as the hematopoietic stem cells were separated respectively. Some of themcontinue to be cultured to identify its properties of stem cell and some of them wereused to extract the RNA to detect the expression and amount of TGF-β and IL-10.Result:1. Morphological observation: Fresh bone marrow mononuclear cells of acutemyeloid leukemia patients are always mixed with a small amount of red blood cells,the cell-size is relatively uniform, round or oval, with strong refraction; Wright’sstaining shows that cell size is relatively even slightly different, with larger nucleusand relatively less cytoplasm; during prolonged incubation time, the cell clone wereformed and increased, and Wright’s stain visibly become difference in cell diameter,the membrane is irregular, creases, with nuclear-mass ratio of different sizes.2. Co-culture:culture system that feeder layer of stromal cells no treated with mitomycin getmore pebble cell formation region.3. Flow cytometry analysis:bone marrow mono-nuclear cells marked by CD34cell ration is dynamic,CD34+CD38-CD123+cells wasproportional to the culture time.4.RT-PCR: mononuclear cells almost totally expressTGF-β mRNA and IL-10mRNA, mRNA expression level in the same cell populationis TGF-β> IL-10.Conclusion:1. Leukemia bone marrow mononuclear cells are easier to form a colony,which mayexist leukemia stem cells.2. experimental study suggests that leukemia stem cells maybe exist in theCD34+CD38-CD123+cell group.3. experimental study suggests that in the primary co-cultured condition stem cellscould be better amplification.4. experimental study suggests that mononuclear cells almost totally express TGF-βmRNA and IL-10mRNA.