| PART ONE Establishment of the model of rat disc chondrocytes apoptosis in vitro Objective To develop an apoptosis model of cartilage endplate using a cellular culture.Methods To mimic the normal internal disc milieu (decreased nutrients and low metabolism), chondrocytes derived from SD rat endplate cartilage were cultured under serum limiting conditions, to select the optimum concentration cells were treated by culture medium with0%,1%,3%,5%,8%,10%fetal bovine serum(FBS)separately Apoptosis was assessed by flow cytometry and by DAPI staining. The expression of apoptosis-related protein was assessed by Western blot.Results Treated with low FBS resulted in changes in cellular morphology-y, DAPI-positive cells increased with the decrese of FBS, apoptosis quantified by flow cytometry also increased significantly (P<0.05) and shown that1%FBS was the optimum concentration;there was strong increased in the expression of apoptosis-related proteins comprise FAS, caspase-3, PARP, cytochrome C(cyt C)in1%FBS group.Conclusion Low serum condition would induce the apoptosis of cartilage endplate chondrocytes.The activation of caspase family would involve in the process. PART TWO Exploration of the apoptosis pathway of intervertebral disc cartilage endplateObjective Cartilage endplate cell apoptosis results in endplate degradation and thinning, which may play a role in the initiation of intervertebral disc degeneration(IVDD). The purpose of this study is to explore the molecular mechanism of disc endplate chondrocytes apoptosis induced by serum starvation.Methods Disc cartilage endplates obtained from3-month old SD rats were subjected to Sequential digestion, chondrocyts were harvest for primary culture. Apoptosis induced by1%fetal bovine serum(FBS) for48hours, chondrocytes were co-incubated with specific caspase-8and-9inhibitors at the same time. Addtionally,10%FBS incubated chondrocytes were taken as control group and co-incubated with DMSO as a positive control.48hours later, apoptosis was detected by DAPI staining and flow cytometery,the expression of caspase-3,-8,-9and their precursors were monitored by western blotting.Result Cellular morphology changed in chondrocytes dealed with low FBS. When quantified by flow cytometery, serum deprivation induced apoptosis in about40.8%, compared to26.1%in unteated group. Specific caspases-9inhibitors significantly reduced starvation-induced apoptosis to26.3%. Caspase-8inhibition had no effect on chondrocyte death(41.3%). Serum deprivation induced caspase-3activation was significantly reduced in caspase-9treatment group.Conclusion These results suggest that serum starvation induced cartilage endplate cells apoptosis is due to caspases activation, predominantly due to caspase-9-dependent pathway and is linked to endplate degeneration and degenerative disc diseases. PART THREE The effect of lentivirus-mediated caspase-3siRNA on intervertebral disc degenerationObjective To investigate the antiapoptotic effects of lentivirus-mediated caspase-3and in intervertebral disc degeneration in rats.Method First we developed a degenerative disc animal model in bipedal rats for the study, lentivirus-mediated caspase-3diluted with PBS was injected into disc for20ul,20ul PBS was ted as control. Animals were killed after fluoroscopic examination, spinal columns were harvested and paraffin embed-ding for histologic and immunohistochemical staining for caspase-3, apoptosis of endplate was detected by TUNEL.Result Caspase-3was down-regulated in the caspase-3siRNA group, fluoroscopic examination and histologic evaluation showed that degenerative changes were significantly suppressed in the caspase3siRNA group. Quantification of TUNEL staining showed that the caspase3siRNA group had significantly fewer apoptotic endplate cells than the control siRNA group.Conclusion These results indicated that down-regulated caspase-3expression can effectively prevent apoptosis of endplate chondrocytes, thus regulating intervertebral disc degeneraton.
|
PDF Full Text Request