营养剥夺诱导髓核细胞Bcl-2/腺病毒干扰蛋白3表达及线粒体转位的实验研究  

UPREGULATION OF Bcl-2/ADENOVIRUS E1B 19-kDa-INTERACTING PROTEIN 3 AND TRANSLOCATION TO MITOCHONDRIA IN NUCLEUS PULPOSUS CELLS INDUCED BY NUTRITION DEPRIVATION

作　　者：刘杰[1,3] 彭娜[2] 曾顺福[1] 陆焱[4] 王建[1] 周跃[1] 

机构地区：[1]第三军医大学新桥医院骨科,重庆400037 [2]第三军医大学新桥医院骨科手术室 [3]解放军93197部队卫生所 [4]解放军第187医院骨科

出　　处：《中国修复重建外科杂志》2012年第2期166-171,共6页Chinese Journal of Reparative and Reconstructive Surgery

基　　金：国家自然科学基金资助项目(30872601)~~

摘　　要：目的通过营养剥夺模拟体内髓核细胞退变微环境,检测Bcl-2/腺病毒干扰蛋白3(Bcl-2/adenovirusE1B 19-kDa-interacting protein 3,BNIP3)表达及线粒体转位情况,为进一步探索髓核细胞退变死亡机制提供实验依据。方法成年清洁级SD大鼠2只,雌雄不限,体重150～200 g。体外分离获取鼠尾椎问盘髓核细胞,将传代后细胞分别置入正常环境(对照组:L-DMEM培养基、10%FBS、21%O_2)和营养剥夺环境(实验组:DMEM无糖无血清培养基、1%O_2)培养24、48、72 h后,实时荧光定量PCR、细胞免疫荧光染色及Western blot检测BNIP3基因及蛋白表达,流式细胞仪检测凋亡率及线粒体膜电位。结果实时荧光定量PCR、细胞免疫荧光染色及Western blot检测显示对照组细胞低表达BNIP3;实验组随培养时间延长,BNIP3表达呈上升趋势,且BNIP3与线粒体相结合;除培养后24 h实验组BNIP3基因表达与对照组比较差异无统计学意义(P>0.05)外,其余各时间点实验组BNIP3基因及蛋白表达与对照组比较差异均有统计学意义(P<0.05)。流式细胞仪检测显示,对照组细胞凋亡率较低,且细胞保持较高的线粒体膜电位;而实验组随培养时间延长细胞凋亡率增加、线粒体膜电位降低,与对照组比较差异均有统计学意义(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线粒体功能障碍,最终导致髓核细胞死亡。Objective To detect the expression of Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 （BNIP3） in cell death induced by nutrition deprivation in nucleus pulposus cells so as to further understand the mechanism of death in nucleus pulposus cells. Methods Two adult sprague dewley rats, male or female, weighing 150-200 g, were involved in this experiment. The cells isolated from rat caudal disc were cultured under the condition of L-DMEM cultrure media, 10%FBS, and 21%O2 （control group） and under the condition of DMEM-free glucose cultrure media, no serum, and 1% O2 （experimental group）. The expressions of BNIP3 gene and protein were detected by real-time fluorescent quantitative PCR, immunofluorescence staining, and Western blot. The cell apoptosis rate and mitochondrial membrane potential were measured by flow cytometry at 24, 48, and 72 hours after culture. Results The expression of BNIP3 decreased in the control group; the expression of BNIP3 showed an increasing tendency with time in the experimental group, and BNIP3 combined with mitochondria. Significant differences were observed in the expressions of BNIP3 gene and protein between 2 groups at the other time （P 〈 0.05） except that no significant difference was observed in the expression of BNIP3 gene at 24 hours （P 〉 0.05）. The cell apoptosis rate and mitochondrial membrane potential were significantly lower in the experimental group than those in the control group （P 〈 0.05）. Conclusion Up-regulation of BNIP3 and translocation to mitochondria may be involved in nucleus pulposus cell death in nutrition deprivation.

关 键 词：髓核细胞 Bcl-2/腺病毒干扰蛋白3 线粒体 营养剥夺 大鼠 

分 类 号：R363[医药卫生—病理学]