大鼠椎间盘髓核细胞体外凋亡模型的建立

Establishment of an apoptosis model of rat disc nucleus pulposus cells in vitro

目的建立大鼠椎间盘髓核细胞体外凋亡模型。方法为了充分模拟退变椎间盘内营养缺乏的微环境,本实验分别采用含1%,3%,5%,8%,10%胎牛血清的DMEM培养基培养椎间盘髓核细胞,筛选最佳促凋亡浓度,分别检测髓核细胞凋亡率、凋亡相关蛋白Bax、bcl-2、caspase-3酶的表达、细胞增殖曲线及免疫荧光分析。结果流式细胞仪测得髓核细胞凋亡率随着胎牛血清(FBS)浓度降低而升高,3%FBS为最有效诱导凋亡浓度;Western blot示Bax、caspase-3酶表达在3%FBS组明显高于10%FBS组,同时bcl-2表达下降;CCK-8检测结果显示含3%FBS的培养基内,随着凋亡率的增长,髓核细胞增殖的速率越来越慢;免疫荧光分析3%FBS组FAS表达量明显比10%FBS组增高。结论 3%FBS能诱导髓核细胞发生凋亡,最终会导致细胞功能丧失,caspase家族参与并执行了这一过程。

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 containing 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 cytometry 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 demonstrated 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.