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.
ABSTRACT
Objective To study the effects of apoptosis-stimulating protein of ρ53-2 (ASPP2) gene on hepatocellular carcinoma (HCC) cell proliferation and apoptosis under starvation and the related mechanism. Methods Lentivirus encoding shRNA against ASPP2 was constructedto knockdown ASPP2 expression in hepatoma cell HepG2. Cell proliferation and apoptosis were observed by transmission electron microscopy, MTS analysis, flow cytometry analysis and morphologic changes. The influence of silencing ASPP2 gene on the proliferation and apoptosis under amino acid-starvation and serum-deprivation culture was observed; autophagy inhibitor 3-methyladenine (3-MA) was added in the experiment so as to detect the involvement of autophagy in the changes induced by ASPP2 down-regulation. Results Transmission electron microscopy showed cytoplasmic accumulation of autophagosomes when ASPP2 was knocked down under amino acid-starvation and serum- deprivation (P<0. 05), with increased GFP-LC3 dots (P<0. 05). MTS analysis showed that silence of ASPP2 gene greatly enhanced the proliferation of HepG2 cells (P<0. 05), which could be inhibited by addition of 3-MA (P<0. 05). Microscope observation showed that silence of ASPP2 gene promoted the anti-apoptotic ability of HepG2 cells, which was reversed by treatment with 3-MA. Early sign of apoptosis was observed in shASPP2 + 3-MA group. Annexin V_PI double staining showed that ASPP2 silence decreased the apoptotic rate ofHepG2 cells from (38±5)% to(15±4)% (P<0. 05), and 3-MA treatment increased it to (36 ±3)% (P<0. 05). Conclusion Down-regulation of ASPP2 expression may facilitate the survival and proliferation of HCC cells through activating autophagy under starvation.