Establishment of an apoptosis model of rat disc nucleus pulposus cells in vitro / 中国实验动物学报

ABSTRACT

Objective To develop an apoptosis model of nucleus pulposus cells in cell culture.Methods To mimic the nutrient-deficient microenvironment of degenerative intervertebral disc,nucleus pulposus cells derived from infant SD rat disc were cultured under serum limiting conditions.Nucleus pulposus cells were cultured in culture medium contai-ning 1%, 3%, 5%, 8%and 10%fetal bovine serum( FBS) respectively to select the optimum FBA concentration.Apoptosis was assessed by flow cytometry, Western blot,cell counting kit, and immunofluorescence technique.Results The flow cy-tometry revealed that apoptosis rate of the nucleus pulposus cells increased with decreasing concentration of FBS, and 3%FBS used in the experimental group was the most effective concentration to induce apoptosis(P<0.05).Western blot dem-onstrated significantly higher expression of Bax and caspase-3 enzyme in the 3%FBS group than in the 10%FBS group, while bcl-2 activity decreased.The results of CCK-8 test indicated that the nucleus pulposus cells got slower proliferation in the medium containing 3%FBS.Immunofluoresence analysis showed that FAS expression was significantly higher in the 3%FBS group than in the 10%FBS group.Conclusions 3%FBS condition may induce apoptosis in the nucleus pulposus cells and compromise the cell function to induce intervertebral disc degeneration.The caspase family should be involved in the process.