골수 단핵구 배양에 대한 조혈 성장인자의 효과

Abstract

Now a days, hematopoietic stem cell transplatation can be used as an effective treatment modality for bone marrow failure syndromes such as aplastic anemia, malignancies, and certain metabolic disease. Hematopoietic stem cells are the population of cells capable both of self renewal and differentiation into a variety of hematopoietic lineages. It is suggested that clinical practice in the areas of bone marrow transplantation and gene therapy might rely on the ex vivo expansion of hematopoietic stem/progenitor cells. The enumeration of cell expression of CD34 and granulocyte macrophage colony forming unit (CFU-GM) are used for assessing the efficacy of human hematopoietic stem cell enrichment technique. The colony stimulating factors (CSF) stimulate the proliferation and differentiation of progenitor cells. We pursued a series of experiments to define the proper conditions for the expansion of hematopoietic stem cells in the short term liquid suspension culture of bone marrow cells and to define optimal combination of colony stimulating factors(CSFs). Liquid cultures were initiated with 1x10 bone marrow mononuclear cells (BM MNCs), and innoculated with 1x10 CD 34 cells, which were isolated from BM MNCs by flow cytometry, in 3 well petri dishes with the various hematopoietic growth factors (HGF). The recombinant human HGFs used are 100ng/mL of stem cell factor (SCF), 100 ng/mL of granulocyte colony-stimulating factor (G-CSF), 100 ng/mL of granulocyte-macrophage colony-stimulating factor (GM-CSF), 100ng/mL of interleukin-3 (IL-3) and combination of SCF 100ng/mL, GM-CSF 100ng/mL, IL-3 100ng/mL. At the end of the culture, colony forming cells were evaluated by semisolid clonogenic assay(CFU-GM) and CD34+ cells were enumerated with flow cytometry. On the third day of the culture, CD34+ cells were expanded 14.98 fold with the addition of SCF, 7.82 fold with G-CSF, 11.12 fold with GM-CSF, 15.53 fold with IL-3 and 18.32 fold with SCF, GM-CSF and IL-3. On the seventh day of the culture, CD34+ cells were expanded 5.82 fold with SCF,5.91 fold with G-CSF,13.94 fold with GM-CSF, 6.43 fold with IL-3 and 9.45 fold with SCF, GM-CSF and IL-3. In CFU-GM assay of the three day culture, CFU-GM counts were 37.5 with SCF, 74.1 with G-CSF, 90.5 with GM-CSF, 126.7 with IL-3, and 125.8 with SCF, GM-CSF and IL-3. The CFU-GM counts of the seven day culture are 31.4 with SCF, 44.6 with G-CSF, 42.3 with GM-CSF, 55.2 with IL-3, and 90.2 with SCF, GM-CSF and IL-3. The three day culture of BM MNCs was more effective than the seven day culture of CD34+ cell expansion and CFU-GM expansion. The results of this study suggest that short term culture of bone marrow cells could expand hematopoietic progenitor cells with the addition of HGFs. Especially, three day culture of BM MNCs with the addition of SCF, GM-CSF and IL-3 might be the most efficient in this system.