中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2011年
6期
983-985
,共3页
朱伟荣%叶伟%徐康%娄振凯%任晋可%陈晋宸%李春海
硃偉榮%葉偉%徐康%婁振凱%任晉可%陳晉宸%李春海
주위영%협위%서강%루진개%임진가%진진신%리춘해
椎间盘退变%纤维环细胞%自噬
椎間盤退變%纖維環細胞%自噬
추간반퇴변%섬유배세포%자서
Intervertebral disc degeneration%Annulus fibrosus cell%Autophagy
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's balanced salt)液饥饿诱导处于对数生长期的纤维环细胞,0、1、2、3 h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Western blot方法分别检测纤维环细胞微管相关蛋白1轻链3(MAPLC3)和Beclin-1 mRNA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;1 h后LC3-Ⅱ及Beclin-1 mRNA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);1 h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.
目的 觀察饑餓誘導對椎間盤纖維環細胞自噬因子LC3和Beclin-1錶達的影響.方法 用EBSS(Earle's balanced salt)液饑餓誘導處于對數生長期的纖維環細胞,0、1、2、3 h後用單丹磺酰戊二胺(MDC)熒光染色法檢測自噬體形成、逆轉錄-聚閤酶鏈反應(RT-PCR)和Western blot方法分彆檢測纖維環細胞微管相關蛋白1輕鏈3(MAPLC3)和Beclin-1 mRNA和蛋白的錶達.結果 隨饑餓時間的延長,MDC暘性細胞數及自噬泡數量增多;1 h後LC3-Ⅱ及Beclin-1 mRNA錶達開始增加,且隨時間增加有增加趨勢(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);1 h後蛋白LC3-Ⅱ/LC3-Ⅰ比值增加,Beclin-1蛋白錶達亦增加,且隨時間增加有增加趨勢(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).結論 饑餓可誘導椎間盤纖維環細胞自噬的髮生,這可能與椎間盤退變的髮生、髮展有關.
목적 관찰기아유도대추간반섬유배세포자서인자LC3화Beclin-1표체적영향.방법 용EBSS(Earle's balanced salt)액기아유도처우대수생장기적섬유배세포,0、1、2、3 h후용단단광선무이알(MDC)형광염색법검측자서체형성、역전록-취합매련반응(RT-PCR)화Western blot방법분별검측섬유배세포미관상관단백1경련3(MAPLC3)화Beclin-1 mRNA화단백적표체.결과 수기아시간적연장,MDC양성세포수급자서포수량증다;1 h후LC3-Ⅱ급Beclin-1 mRNA표체개시증가,차수시간증가유증가추세(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);1 h후단백LC3-Ⅱ/LC3-Ⅰ비치증가,Beclin-1단백표체역증가,차수시간증가유증가추세(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).결론 기아가유도추간반섬유배세포자서적발생,저가능여추간반퇴변적발생、발전유관.
Objective To investigate the starvation-induced changes of LC-3 and Beclin-1 expression in annulus fibrosus cells. 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 monodansylcadaverine ( MDC) fluorescent staining method. Reverse transcription-polymerase chain reaction ( RT-PCR) and Western blotting analyses were used to detect the expression of microtubuleassociated 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 ±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). The ratio of LC3-Ⅱ/LC3-Ⅰ and Beclin-1 protein expression was also increased with the prolongation of starvation (LC3-Ⅱ/LC3- Ⅰ : 0. 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.
