饥饿诱导对纤维环细胞自噬因子LC3和Beclin-1表达的影响  

Starvation-induced changes of LCo3 and Beclin-1 expression in annulus fibrosus cells

作　　者：朱伟荣[1] 叶伟[1] 徐康[2] 娄振凯[1] 任晋可[1] 陈晋宸[3] 李春海[1] 

机构地区：[1]中山大学孙逸仙纪念医院脊柱外科,广州510120 [2]中山大学孙逸仙纪念医院普外科实验室,广州510120 [3]中山大学

出　　处：《中华实验外科杂志》2011年第6期983-985,共3页Chinese Journal of Experimental Surgery

基　　金：基金项目：教育部博士点新教师基金资助项目（20090171120077）;广东省自然科学基金资助项目（10151008901000084）;广东省科技计划基金资助项目（20088060600059,20098060700097）;广东省医学科学基金资助项目（A2009188）;广东省大学生创新实验项目（1055810096）

摘　　要：目的观察饥饿诱导对椎间盘纤维环细胞自噬因子LC3和Beclin-1表达的影响。方法用EBSS（Earle’sbalancedsalt）液饥饿诱导处于对数生长期的纤维环细胞，0、1、2、3h后用单丹磺酰戊二胺（MDC）荧光染色法检测自噬体形成、逆转录－聚合酶链反应（RT—PCR）和Westernblot方法分别检测纤维环细胞微管相关蛋白1轻链3（MAPLC3）和Beclin－1mRNA和蛋白的表达。结果随饥饿时间的延长，MDC阳性细胞数及自噬泡数量增多；1h后LC3－II及Beclin－1mRNA表达开始增加，且随时间增加有增加趋势（LC3—II：0．21±0．02、0．45±0．12、0．60±0．05、0．74±0．03，P〈0．05；Beclin-1：0．20±0．06、0．37±0．11、0．52±0．07、0．69±0．07，P〈0．05）；1h后蛋白LC3－II／LC3－I比值增加，Beclin．1蛋白表达亦增加，且随时间增加有增加趋势（LC3－II／LC3－I：0．12±0．05、0．32±0．09、0．63±0．07、0．92±0．04，P〈0．01；Beclin－1：0．61±0．10、0．83±0．11、1．09±0．05、1．35±0．10，P〈0．05）。结论饥饿可诱导椎间盘纤维环细胞自噬的发生，这可能与椎间盘退变的发生、发展有关。Objective To investigate the starvation-induced changes of LC-3 and Beclin-1 expression in annulus fibrosus ceils. Methods Rat annulus fibrosus cells in the logarithmic growth phase were cultured with amino acid free EBSS （Earle＇ s balanced salt） medium instead of the DMEM-F12 medium. Cells were collected at 1, 2 and 3 h after EBSS culture. Autopagosomes in annulus fibrosus cells were detected with monodausylcadaverine （MDC） fluorescent staining method. Reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting analyses were used to detect the expression of microtubule- associated protein 1 light chain 3 （LC3） and Beclin-1 in annulus fibrosus cells. Results Compared with the control group, MDC positive cells and autopagosomes were increased with the prolongation of starvation. LC3- II and Beclin-1 mRNA expression levels in annulus fibrosus cells were increased with the prolongation of starvation （ LC3- II ： 0. 21 ± 0. 02, 0.45 ± 0. 12, 0. 60 ± 0. 05, 0.74 ± O. 03, P 〈 0. 05 ; Beclin-1 ： 0. 20 ±0. 06, 0. 37 ±0. 11, O. 52 ±0. 07, 0. 69 ±0.07 ,P 〈0. 05）. The ratio of LC3- II/LC3- I and Beelin-1 protein expression was also increased with the prolongation of starvation （LC3-II/LC3- I ： O. 12 ± 0.05, 0.32±0.09, 0.63 ±0.07, 0. 92±0. 04,P 〈0. 05; Beclin-1 ： 0.61 ±0. 10, 0.83 ±0. 11, 1.09 ±0.05,1. 35 ± 0. 10,P 〈 0.05 ）. Conclusion Autophagy of annulus fibrosus cells can be induced by starvation, which may be related to the procession of intervertebral disc degeneration.

关 键 词：椎间盘退变 纤维环细胞 自噬 

分 类 号：R363[医药卫生—病理学]