摘要目的 通过营养剥夺(低氧-无糖-无血清)诱导髓核细胞凋亡,检测髓核细胞线粒体蛋白BNIP3(bcl-2/Adenovirus El B19-kd Protein-Interacting Protein 3)表达。方法 体外分离培养大鼠尾椎间盘髓核细胞,传代至P1代后将细胞分为正常对照组:DMEM/L培养基(1000 mg葡萄糖/L)、10％胎牛血清、21％ O2;实验组:DMEM无糖培养基、无血清、1％ O2;分别将两组细胞培养0、24、48、72 h后,免疫荧光检测BNIP3蛋白表达并定位、流式细胞仪检测细胞凋亡率及线粒体膜电位。结果 免疫荧光检测显示正常对照组细胞低表达BNIP3,定位于胞质且未结合线粒体,实验组显示随培养时间延长,BNIP3表达呈上升趋势,在72 h明显增加,共定位发现BNIP3主要分布于胞质,并与线粒体相结合;流式细胞仪检测发现正常对照组细胞凋亡率为(1.02±0.21)％,实验组细胞凋亡率明显增加(P＜0.05),分别为(4.22±0.34)％、(7.5l±2.33)％、( 19.93±2.58)％,线粒体膜电位结果显示实验组24、48、72 h线粒体膜电位均不同程度降低,分别为(90.23±1.32)％、(87.30±2.21)％、(82.63±2.91)％,均显著低于正常对照组(94.21±3.96)％(P＜0.05)。结论 营养剥夺可能通过诱导BNIP3表达增加并结合线粒体导致髓核细胞凋亡。

abstractsObjective To detect the expression of BNIP3 and apoptosis induced by nutrition deprivation in nucleus pulposus cells. Methods Rat caudal disc nucleus pulposus cells were harvested and passaged to P1, and divided into two groups: control group (cells cultured in DMEM/L medium, 21％O2,10％ FBS) ; experimental group ( cells cultured in DMEM with no glucose, no serum, 1％ O2 ). After cells were cultured for 0, 24, 48 and 72 h, the expression of BNIP3 was detected by using immunofluorescence staining, and apoptosis rate and mitochondrial membrane potential were measured by using flow cytometry.Results Immunofluorescence staining showed that BNIP3 proteins in control group were expressed in cytoplasm with low level and did not integrate with mitochondria, but in experimental group, higher expression was found during culture in nutrition deprivation, especially in 72 h, furthermore, the proteins integrated with mitochondria. The apoptosis rate in control group was ( 1.02 ± 0. 21 ) ％ , and that in experimental group was (4. 22 ±0. 34)％, (7.51 ±2. 33)％ and (19. 93 ±2. 58)％ at 24, 48 and 72 h respectively.The mitochondrial membrane potential was significantly reduced in experimental group to varying degrees [(90. 23 ± 1.32)％, (87.30 ±2. 21 )％ and (82. 63 ±2. 91 )％ at 24, 48 and 72 h respectively], as compared with control group (94. 21 ± 3.96) ％ ( P ＜ 0. 05). Conclusion Nutrition deprivation may upregulate the BNIP3 expression to mediate apoptosis in nucleus pulposus cells.