[The effect of down regulating of p27 on the activity of hematopoietic progenitor cells derived from umbilical cord blood and bone marrow ex vivo].

OBJECTIVE

To compare p27 interfering effect on the proliferation and hematopoietic potential of hematopoietic progenitor cells (HPC) derived from human bone marrow (BM) and umbilical cord blood (UCB), and investigate the related mechanism.

METHODS

CD34(+) cells sorted from human BM by flow cytometry and isolated from UCB by a magnetic isolation system were infected with retrovirus-p27 antisense cDNA (p27AS) and cultured with cocktail-cytokines. The cell proliferation capacities were detected by cell growth curve and DNA content analysis, and the hematopoietic potential by colony formation assay. The protein expression of p27 and CDK2 were measured with Western blot. CD34(+) cells infected with retrovirus-p27 sense cDNA (p27SE) and virus-green fluorescence protein (GFP) were used as the control.

RESULTS

Comparing with groups of GFP and p27SE, p27AS showed to accelerate the expansion of UCB HPC significantly (P < 0.01), the cell number of p27AS, p27SE and GFP increased by 197.3 +/- 47.7-, 12.7 +/- 8.1- and 41.8 +/- 30.6- fold respectively by the end of the 9-day culture, the BM HPC increased by 36.0 +/- 22.3-, 8.7 +/- 6.8- and 14.1 +/- 10.4-fold respectively in the same time as UCB HPC. Cell cycle analysis showed p27AS mainly promoted S phase of BM and UCB HPC. S phase cell percentages of UCB HPC infected with p27AS, p27SE and GFP were (17.0 +/- 4.8)%, (2.0 +/- 0.8)% and (4.1 +/- 1.8)% and that of BM HPC were (8.4 +/- 4.4)%, (1.0 +/- 0.7)% and (3.8 +/- 1.4)% respectively. The yields of colony formation of p27AS for BM and UCB was higher than that for GFP (P < 0.05). Western blot demonstrated the expression of p27 reduced in the p27AS group, while CDK2 increased in the group. The virus infection rate, cell growth rate and colony forming yields of BM HPC was inferior to that of UCB HPC.

CONCLUSIONS

p27 gene interfering could promote HPC proliferation, hematopoietic potential and the response to stimulations of UCB HPC is higher than that of BM HPC ex vivo. It seems that cell cycle controlled by p27 was related to CDK2.