中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2013年
2期
354-357
,共4页
马凯歌%邵增务%王佰川%熊蠡茗%吴强%杨述华
馬凱歌%邵增務%王佰川%熊蠡茗%吳彊%楊述華
마개가%소증무%왕백천%웅려명%오강%양술화
椎间盘%自噬%压力%髓核细胞
椎間盤%自噬%壓力%髓覈細胞
추간반%자서%압력%수핵세포
Intervertebral disc%Autophagy%Compression%Nucleus pulposus cells
目的 观察自噬是否参与压力诱导兔髓核细胞退变,探讨压力诱导髓核细胞退变机制.方法 从3月龄兔胸腰段脊柱提取兔髓核细胞培养,取第2代兔髓核细胞,1 Mpa压力环境下培养12、24、48 h.细胞活力与细胞毒性检测观察压力对细胞生长的影响,透射电镜观察压力条件下细胞的微观结构,单丹磺酰尸胺(MDC)染色观察细胞内自噬性空泡结构及自噬率,实时荧光定量聚合酶链反应(FQ-PCR)观察自噬相关基因在压力条件下的表达.结果 压力导致细胞衰老及死亡,透射电镜见细胞内存在自噬体结构,MDC染色后行流式细胞学检测显示12、24、48 h自噬率分别为(1.580±0.171)％、(7.930±0.252)％、(13.530±1.206)％,PCR结果显示压力诱导自噬相关基因(LC3B、Beclin-1)表达量与正常条件下表达明显增多(P＜0.05),且随着压力培养时间延长,LC3B、Beclin-1表达量也明显增多(P＜0.05).结论 自噬参与压力诱导细胞退变的过程,为压力诱导椎间盘髓核细胞退变的防治提供新的途径.
目的 觀察自噬是否參與壓力誘導兔髓覈細胞退變,探討壓力誘導髓覈細胞退變機製.方法 從3月齡兔胸腰段脊柱提取兔髓覈細胞培養,取第2代兔髓覈細胞,1 Mpa壓力環境下培養12、24、48 h.細胞活力與細胞毒性檢測觀察壓力對細胞生長的影響,透射電鏡觀察壓力條件下細胞的微觀結構,單丹磺酰尸胺(MDC)染色觀察細胞內自噬性空泡結構及自噬率,實時熒光定量聚閤酶鏈反應(FQ-PCR)觀察自噬相關基因在壓力條件下的錶達.結果 壓力導緻細胞衰老及死亡,透射電鏡見細胞內存在自噬體結構,MDC染色後行流式細胞學檢測顯示12、24、48 h自噬率分彆為(1.580±0.171)％、(7.930±0.252)％、(13.530±1.206)％,PCR結果顯示壓力誘導自噬相關基因(LC3B、Beclin-1)錶達量與正常條件下錶達明顯增多(P＜0.05),且隨著壓力培養時間延長,LC3B、Beclin-1錶達量也明顯增多(P＜0.05).結論 自噬參與壓力誘導細胞退變的過程,為壓力誘導椎間盤髓覈細胞退變的防治提供新的途徑.
목적 관찰자서시부삼여압력유도토수핵세포퇴변,탐토압력유도수핵세포퇴변궤제.방법 종3월령토흉요단척주제취토수핵세포배양,취제2대토수핵세포,1 Mpa압력배경하배양12、24、48 h.세포활력여세포독성검측관찰압력대세포생장적영향,투사전경관찰압력조건하세포적미관결구,단단광선시알(MDC)염색관찰세포내자서성공포결구급자서솔,실시형광정량취합매련반응(FQ-PCR)관찰자서상관기인재압력조건하적표체.결과 압력도치세포쇠로급사망,투사전경견세포내존재자서체결구,MDC염색후행류식세포학검측현시12、24、48 h자서솔분별위(1.580±0.171)％、(7.930±0.252)％、(13.530±1.206)％,PCR결과현시압력유도자서상관기인(LC3B、Beclin-1)표체량여정상조건하표체명현증다(P＜0.05),차수착압력배양시간연장,LC3B、Beclin-1표체량야명현증다(P＜0.05).결론 자서삼여압력유도세포퇴변적과정,위압력유도추간반수핵세포퇴변적방치제공신적도경.
Objective To investigate the involvement of autophagy in the progression of compression-induced intervertebral disc (IVD) degeneration.Methods Rabbit nucleus pulposus (NP) cells were isolated from the thoracolumbar IVD of 3-month-old Japanese white rabbits.The rabbit NP cells at the second generation were cultured under the compression condition of 1 Mpa for 12,24 or 48 h.Cell viability was determined by using cell counting Kit-8 (CCK8).The ultrastructural features of rabbit NP cells exposed to compression were examined under the transmission electron microscopy (TEM).The presence of autophagic vacuoles,as a marker of autophagy,was detected by using fluorescent dye monodansylcadaverine (MDC).The variation of autophagy-related gene expression was analyzed by using real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) to evaluate the effect of compression on autophagy of rabbit NP cells.Results Compression induced rabbit NP cell degeneration and death.The autophagosomes were detected in rabbit NP cells under the TEM.The autophagic rate was (1.580 ± 0.171) ％,(7.930 ±0.252) ％,(13.530 ± 1.206) ％ at 12,24,and 48 h respectively.The mRNA expression of beclin-1 and LC3B was significantly higher in the compression treatment group than in the control group (P ＜ 0.05),and the mRNA expression of beclin-1 and LC3B was increased over time under compression culture condition.Conclusion Autophagy and autophagic cell death were present in compression-induced rat NP cell injury,which offers a novel sight about compression-induced degeneration of IVD cells.
