Expression Of Proliferating Cell Nuclear Antigen In Cord Blood CD34～+ Hematopoietic Stem/Progentior Cells And Its Significance

Human cord blood is a rich source of hematopoiectic stem/progenitor cells. It has been proved to be a alternative source of stem cell besides bone marrow and peripheral blood. Till now cord blood transplantion has been widely used in treating leukemia and malignant tumor in children. However, one of the main limiting factors for increased use of cord blood in adult allogeneic transplantation is the small number of progenitor cells that can be collected and infused. Ex vivo expansion of cord blood might help to overcome this limitation. Hematopoiectic stem/progenitor cells from cord blood have a large proliferative capacity on the stimulation of cytokines, but also lose selfe-renew activity. Therefore, in resent years, many studies has been carried out with the aim of establishing a combination cytokines that would allow ex vivo expansion of the more primitive haematopoietic progenitor cells, but , despite this concerted effort, no definitive agreement on a common expansion protocol has so far been reached. In contrast to the large body of studies on the protocol of cytokines combination, less information is known about cell cycle regulation of cord blood progenitor cells. As known to all, cell proliferation and differentiation are controlled by the cell cycle. Hematopoietic stem cells proliferation and differention is closely associated with cell cycle regulation factors.Proliferating cell nuclear antigen(PCNA) is one important cell cycleregulation proteins, driving cells into S-phase. In order to reveal the significance of PCNA on cell cycle regulation , we use flow cytometer to detect PCNA level and cell cycle in cord blood CD34+ cells in different condition during different culture time.1.PCNA expression level in cord blood CD34+ cells during ex vivo culture.CD34+ cells were isolated by Mini MACS(magnetic activated cell sorting), and then cultured in IMDM medium with different growth factors combination ( (1) no growth factor ; (2) SCF+IL-6+IL-3 ; (3) SCF+IL-6+IL-3+Tpo ; @ FL+Tpo+SCF + IL-6+IL-3) . On day 3 , 5, 7 of incubation, cells were harvested, and analyzed for PCNA protein level by flow cytometer. The results have shown that PCNA protein was lowly expressed in freshly isolated cord blood CD34+ cells, the positive rate was (11.6+5.2 ) %, in vitro culture of the cells, PCNA protein was dropped down gradually without growth factor , while increased significantly in the presense of vaious growth factor, especially the combination of SCF, IL-3JL-6, FL and Tpo. After 7days culture, its protein level reached (78.2+8.7) %. 2.cell cycle status in cord blood CD34+ cells during ex vivo expansion.CD34+ cells were isolated by Mini MACS, and then cultured in IMDM medium with different growth factors combination ((1)no growth factor; (2) SCF+IL-6+IL-3; (3)SCF+IL-6+IL-3+Tpo; (4)FL+Tpo+SCF + IL-6+IL-3) .In different culture time(0d 3d 7d), cells were collected, stained with propidiumiodide(PI), and analyzed for cell cycle by flow cytometer. Results show that 95.1% of freshly isolated cord blood CD34+ cells were in GO/Gl phase and only 4.9% in S phase. On day 3 of vitro culture in the presence of various growth factor, cells were drived into cell cycle, on day 7, the number of S/G2/M phase cells was increase significantly.The studies suggest that in the presence of various growth factor, PCNA protein in cord blood CD34+ cells was increased significantly, and drive cells into the S phase.