2014 Volume 23 Issue 3 Pages 343-350
The objectives of this study were to screen for the better Oligonucleotide (ODN) that promotes the proliferation of human bone-marrow mesenchymal stem cells (hBMSCs) and to investigate the mechanism of action of the ODN that has the greatest effect on the hBMSCs cell cycle. hBMSCs were isolated, cultured to the third passage and subjected to osteogenic, adipogenic and chondrogenic induction in order to examine their capacities for multi-differentiation. The hBMSCs were seeded at different plating densities (3.0×103/cm2, 6.0×103/cm2, 1.2×104/cm2, 2.4×104/cm2) and tested for seven consecutive days to determine the better plating density. A total of 12 experimental groups and 1 control group of hBMSCs (4 replicate wells in each group) were established and treated with ODN types MT01, FC003, or SAT05f at concentrations of 0.5mg/l, 1.0 mg/l, 2.0 mg/l or 4.0 mg/l; the control group was treated with an equal volume of PBS. Proliferation of hBMSCs was determined for 3 consecutive days after treatment via CCK-8 assay. The type and concentration of ODN that had a significant facilitatory effect on hBMSCs proliferation was selected and cell cycle analysis was done on days 1, 2 and 3 after ODN treatment; control groups were treated with an equal amount of PBS. The expressions of cyclin A, cyclin D1, cyclin dependent kinase (CDK) 2 and CDK 4 in hBMSCs were measured on day 2 after treatment using fluorescent quantitative real-time PCR. The isolated and cultured hBMSCs were found to have osteogenic, adipogenic and chondrogenic differentiation capacities. The better cell growth curve was found to occur at a plating density of 6.0×103/cm2. Optical density was significantly increased in hBMSCs treated with 0.5 mg/L FC003 on day 1 (P<0.01) compared to the control group. Optical density was significantly decreased on day 1 after treatment with 1.0 mg/L and 4.0 mg/l of SAT05f (P<0.01 and P<0.05, respectively). Optical density was significantly increased on days 1, 2 and 3 after treatment with a final concentration of 2.0 mg/L of MT01 (P<0.01, P<0.05, P<0.05, respectively). The percentage of cells in phase G0/G1 was significantly reduced, and the percentage of cells in phases S and G2/M was significantly increased (P<0.01) after treatment with 2.0 mg/l MT01 compared to the control group. Furthermore, the expressions of cyclin A, cyclin D1, CDK 2 and CDK 4 were significantly elevated (P<0.01) compared with the control group. In conclusion, a 2.0 mg/l concentration of MT01 significantly promotes hBMSCs proliferation as evidenced by the decrease in the percentage of cells in phase G0/G1 and the increase in the percentage of cells in phases S and G2/M. The underlying molecular mechanisms may include, but are not limited to, elevated expressions of cyclin A, cyclin D1, CDK 2 and CDK 4.
