| Backgroud and objecticv: Human cord blood is a rich source of hematopotic 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 transplantation has been widely used in treating children leukemia, inborn immune deficiency, heritage metabolic disease and so on. However, one of the main limiting factor for increased use of cord blood allogeneic transplantation in adult and adolescent is the small number of progenitor cells that can be collected and infused. Ex vivo expansion of cord blood is developing very fast, might help to overcome this limitation. Hematopoietic stem / progenitor cells from cord blood have a large proliferative capacity on the stimulation of cytokines, but also become maturation and lose self-renew activity. Therefore, in resent years, many studies has been carried out with the aim of establishing a combination cytokines that could allow ex vivo expansion of the more primitive hematopoietic 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 studied on the protocol of cytokines combination, less information is known about cell cycle regulation of cord blood progenitor cells. Not only relationship with cytokines, hematopoietic stem cells proliferationand differentiation is also closely associated with cell cycle regulation factors. Proliferation cell nuclear antigen(PCNA) is one important cell cycle regulation proteins, driving cells into S-phase. In order to improve the possibility of control the cell cycle regulation proteins in ex vivo expansion of hematopotic stem / progenitor cells, we study the effect of proliferating cell nuclear antigen antisense oligonucleotide on ex vivo expansion of cord blood CD34+ hematopoietic stem/progenitor cells.Methods: CD34+cells were purified from fresh cord blood by immunomagnetic beads, CD34+cells were incubated in liquid culture system with growth factors combination(SCF+ IL-6+IL-3+Tpo+FL). We divided into 3 groups: ?control group: without PCNA-ASODN ; ?low concentration group: add 0.02umol/ml PCNA-ASODN; ?high concentration group: add 0.03umol/ml PCNA-ASODN. After CD34+cells incubated 7 days, we count the number of nuclear cells; the percentage ofdifferent kinds of stem/progenitor cells, PCNA protein level and cell cycle were measured by flow cytometry.Results: Compared with the control group, PCNA were lowly expressed in tow experiential group, with a positive rate was (27.2 + 3.6)% and(19.0±1.5)%, the difference between the three groups is high significant. The nuclear cells and CD34+cells were expanded weakly in high concentration group, just expanded (7.1 ± 1.6) folds and (2.9 + 0.6) folds. In low concentration group, the nuclear cells and CD34+cells were expanded (15.7 + 1.5) folds and ( 6.7+2.3 ) folds, hardly no difference to control gruop; but the percentage of CD34+cells were increased to (33.4 ±3.2%) , the percentage of CD34+CD38~ were (83.0+2.1) % inall of them, CD34+CD38 cells were expanded (57.8 ±9.9) folds, it has high significant difference compared with the control group. In addition, the number of Go/Gi phase cells in low concentration group is much more than that in control group; but most of the cells in high concentration group is in Go/Gi phase because of low ex vivo expansion.Conclusion: Low concentration PCNA-ASODN can decrease PCNA expressed effectively and slow down differentiation of CD34+cells during ex vivo expansion procedure, improve the expansion efficiency. Our study demonstrate it is impossible to control the cell cycle regulation proteins during the ex vivo expansion of hematopoietic stem / progenitor cells. |
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