[AN IN VITRO STUDY ON BIOLOGICAL CHARACTERISTICS OF BONE MARROW MESENCHYMAL STEM CELLS IN MICROENVIRONMENT OF PREMATURE SENESCENCE OF NUCLEUS PULPOSUS CELLS]

Abstract

Objective: To investigate the biological characteristics of bone marrow mesenchymal stem cells (BMSCs) in microenvironment of premature senescence of nucleus pulposus cells (NPCs) so as to lay a foundation for the repair of intervertebral disc degeneration by BMSCs transplantation.

Methods: Human degenerative nucleus pulposus and normal bone marrow were collected, and then NPCs and BMSCs were isolated, cultured, and identified. The 3rd passage BMSCs and the 1st passage NPCs with premature senescence were co-cultured without contact in the Transwell culture system. NPCs to BMSCs ratio was 75%:25% (group A), 50%:50% (group B), and 0:100% (group C). The morphological changes of BMSCs were observed by inverted phase contrast microscopy and transmission electron microscopy. At 3 and 6 days after co-culture, cell counting kit 8 was used to detect cell viability, flow cytometry was used to observe the cell cycle and detect DNA metabolism after BrdU labeling. Cell senescence was also evaluated by detecting senescence associated β-galactosidase (SA-β- gal) activity.

Results: The typical morphology of cell senescence was seen in groups A and B, especially in group A. At 3 and 6 days after co-culture, the cell survival rate of group A was significantly lower than that of group B (P < 0.05). At 3 days after co-culture, the proportion of cells in G1 phase in group A was significantly higher than that in groups B and C (P < 0.05), the proportion of cells in S phase in group A was significantly lower than that in groups B and C (P < 0.05). At 6 days, the proportion of cells in G1 phase in group A was about 81.0%, and the proportion of cells in S phase and G2 phase decreased, showing significant difference when compared with groups B and C (P < 0.05); the proportion of cells in G phase in group B was about 74.4%, showing significant difference when compared with group C (P < 0.05). BrdU content in group A was significantly lower than that in groups B and C at 3 and 6 days after co-culture (P < 0.05), but no significant difference was found between groups B and C at 3 days (P > 0.05); BrdU content in group B was also significantly reduced when compared with group C (P < 0.05) at 6 days. At 6 days, SA-β-gal activity was significantly increased in groups A and B, and significant difference was shown in SA-β-gal positive cell number between groups (P < 0.05).

Conclusion: Premature senescence of NPCs can down-regulate the proliferation capacity of co-cultured BMSCs by the paracrine effect. The greater proportion of NPCs with premature senescence is, the earlier senescence of BMSCs will be induced.