中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
5期
1087-1090
,共4页
潘峰%祝成亮%程艳香%李章华%陶凤华%陶海鹰%贺斌%余铃%戢鹏
潘峰%祝成亮%程豔香%李章華%陶鳳華%陶海鷹%賀斌%餘鈴%戢鵬
반봉%축성량%정염향%리장화%도봉화%도해응%하빈%여령%집붕
脊髓缺血%他克莫司%活性氧簇%丝氨酸-苏氨酸蛋白激酶
脊髓缺血%他剋莫司%活性氧簇%絲氨痠-囌氨痠蛋白激酶
척수결혈%타극막사%활성양족%사안산-소안산단백격매
Spinal cord ischemia%Tacrolimus%Reactive oxygen species%Serine-threonine protein kinase
目的 探讨他克莫司后处理对缺血再灌注脊髓组织中丝氨酸-苏氨酸蛋白激酶(Akt)信号途径的影响及活性氧簇(ROS)在其中的意义.方法 采用Taira法制备脊髓缺血再灌注损伤模型.雄性SD大鼠80只随机分为4组:假手术组(SO组)、缺血再灌注组(IR组)、他克莫司后处理组(TP组)、缺血后处理+氧自由基清除剂N-乙酰半胱氨酸组(TP+N组).IR组在脊髓缺血20 min后再灌注;TP组在再灌注即刻,经左颈总动脉一次性注射他克莫司0.5 mg/kg,余操作同IR组;TP+N组在脊髓缺血前5min经左颈总动脉注射N-乙酰半胱氨酸150 mg/kg,余操作同TP组;SO组仅行股动脉置管.再灌注15 min取材,采用2',7'-二氯荧光素乙酰乙酸盐(DCFH-DA)荧光分光光度法观察ROS的生成水平;运用Western blot法观察磷酸化Akt(p-Akt)表达水平.再灌注24 h截取相应脊髓节段,应用流式细胞术检测神经细胞凋亡.再灌注7d取材,行神经元尼氏染色观察病理学变化.结果 SO组和再灌注15 min时IR、TP、TP+N组大鼠脊髓组织ROS水平分别为49.0±3.5、145.3±11.7、79.0±6.3和12.8 ±0.9,TP组ROS水平明显低于IR组(P<0.01),TP +N组ROS水平显著低于其他3组(P<0.01).SO组和再灌注15 min时IR、TP、TP+N组大鼠脊髓组织p-Akt表达分别为0.54±0.06、0.34±0.03、0.42 ±0.07和0.28±0,05,IR组p-Akt表达显著低于SO组(P<0.01),TP组明显高于IR组(P<0.05),TP+N组显著低于SO组(P<0.01)和TP组(P<0.01).SO组和再灌注24 h时IR、TP、TP+N组大鼠脊髓组织凋亡率分别为(7.5±1.8)%、(31.4±5.5)%、(22.3±4.0)%和(33.3±3.1)%,IR组和TP+N组凋亡率均明显高于TP组(P<0.01),IR组和TP+N组之间差异无统计学意义(P>0.05).再灌注7d时脊髓组织切片显示SO组无明显病理改变,TP组病理变化较轻,IR组和TP+N组病变严重.结论 他克莫司后处理通过对Akt信号途径的正向调控发挥脊髓保护效应,ROS是启动该机制不可或缺的信号分子.
目的 探討他剋莫司後處理對缺血再灌註脊髓組織中絲氨痠-囌氨痠蛋白激酶(Akt)信號途徑的影響及活性氧簇(ROS)在其中的意義.方法 採用Taira法製備脊髓缺血再灌註損傷模型.雄性SD大鼠80隻隨機分為4組:假手術組(SO組)、缺血再灌註組(IR組)、他剋莫司後處理組(TP組)、缺血後處理+氧自由基清除劑N-乙酰半胱氨痠組(TP+N組).IR組在脊髓缺血20 min後再灌註;TP組在再灌註即刻,經左頸總動脈一次性註射他剋莫司0.5 mg/kg,餘操作同IR組;TP+N組在脊髓缺血前5min經左頸總動脈註射N-乙酰半胱氨痠150 mg/kg,餘操作同TP組;SO組僅行股動脈置管.再灌註15 min取材,採用2',7'-二氯熒光素乙酰乙痠鹽(DCFH-DA)熒光分光光度法觀察ROS的生成水平;運用Western blot法觀察燐痠化Akt(p-Akt)錶達水平.再灌註24 h截取相應脊髓節段,應用流式細胞術檢測神經細胞凋亡.再灌註7d取材,行神經元尼氏染色觀察病理學變化.結果 SO組和再灌註15 min時IR、TP、TP+N組大鼠脊髓組織ROS水平分彆為49.0±3.5、145.3±11.7、79.0±6.3和12.8 ±0.9,TP組ROS水平明顯低于IR組(P<0.01),TP +N組ROS水平顯著低于其他3組(P<0.01).SO組和再灌註15 min時IR、TP、TP+N組大鼠脊髓組織p-Akt錶達分彆為0.54±0.06、0.34±0.03、0.42 ±0.07和0.28±0,05,IR組p-Akt錶達顯著低于SO組(P<0.01),TP組明顯高于IR組(P<0.05),TP+N組顯著低于SO組(P<0.01)和TP組(P<0.01).SO組和再灌註24 h時IR、TP、TP+N組大鼠脊髓組織凋亡率分彆為(7.5±1.8)%、(31.4±5.5)%、(22.3±4.0)%和(33.3±3.1)%,IR組和TP+N組凋亡率均明顯高于TP組(P<0.01),IR組和TP+N組之間差異無統計學意義(P>0.05).再灌註7d時脊髓組織切片顯示SO組無明顯病理改變,TP組病理變化較輕,IR組和TP+N組病變嚴重.結論 他剋莫司後處理通過對Akt信號途徑的正嚮調控髮揮脊髓保護效應,ROS是啟動該機製不可或缺的信號分子.
목적 탐토타극막사후처리대결혈재관주척수조직중사안산-소안산단백격매(Akt)신호도경적영향급활성양족(ROS)재기중적의의.방법 채용Taira법제비척수결혈재관주손상모형.웅성SD대서80지수궤분위4조:가수술조(SO조)、결혈재관주조(IR조)、타극막사후처리조(TP조)、결혈후처리+양자유기청제제N-을선반광안산조(TP+N조).IR조재척수결혈20 min후재관주;TP조재재관주즉각,경좌경총동맥일차성주사타극막사0.5 mg/kg,여조작동IR조;TP+N조재척수결혈전5min경좌경총동맥주사N-을선반광안산150 mg/kg,여조작동TP조;SO조부행고동맥치관.재관주15 min취재,채용2',7'-이록형광소을선을산염(DCFH-DA)형광분광광도법관찰ROS적생성수평;운용Western blot법관찰린산화Akt(p-Akt)표체수평.재관주24 h절취상응척수절단,응용류식세포술검측신경세포조망.재관주7d취재,행신경원니씨염색관찰병이학변화.결과 SO조화재관주15 min시IR、TP、TP+N조대서척수조직ROS수평분별위49.0±3.5、145.3±11.7、79.0±6.3화12.8 ±0.9,TP조ROS수평명현저우IR조(P<0.01),TP +N조ROS수평현저저우기타3조(P<0.01).SO조화재관주15 min시IR、TP、TP+N조대서척수조직p-Akt표체분별위0.54±0.06、0.34±0.03、0.42 ±0.07화0.28±0,05,IR조p-Akt표체현저저우SO조(P<0.01),TP조명현고우IR조(P<0.05),TP+N조현저저우SO조(P<0.01)화TP조(P<0.01).SO조화재관주24 h시IR、TP、TP+N조대서척수조직조망솔분별위(7.5±1.8)%、(31.4±5.5)%、(22.3±4.0)%화(33.3±3.1)%,IR조화TP+N조조망솔균명현고우TP조(P<0.01),IR조화TP+N조지간차이무통계학의의(P>0.05).재관주7d시척수조직절편현시SO조무명현병리개변,TP조병리변화교경,IR조화TP+N조병변엄중.결론 타극막사후처리통과대Akt신호도경적정향조공발휘척수보호효응,ROS시계동해궤제불가혹결적신호분자.
Objective To explore the influence of tacrolimus postconditioning (TP) on serine-threonine protein kinase (Akt) signaling pathway afier spinal cord ischemia-reperfusion injury (SCIRI)and the role of reactive oxygen species (ROS) in the mechanism.Methods The rat model of SCIRI was prepared on the basis of Taira' s method.Eighty male SD rats were randomly divided into 4 groups:sham operation (SO) group,ischemia-reperfusion (IR) group,TP group,and TP plus N-Acetylcysteine (TP +N) group.IR group underwent reperfusion for 20 min after spinal cord ischemia.TP group experienced a single injection of tacrolimus (0.5 mg/kg) through the left common carotid artery right at the onset of reperfusion,with remaining procedures the same as IR group.TP + N group was injected through the left common carotid artery with N-Acetylcysteine (150 mg/kg) 5 min before spinal cord ischemia,with remai-ning procedures the same as TP group.SO group received exclusively femoral artery catheterization.Fluo-rospectrophotometry was employed to detect the level of ROS in injured spinal cord segments,and immuno-blotting was used to determine the expression of phosphorylated Akt (p-Akt) at 15 min after reperfusion.Flow cytometry was applied to assess apoptosis of neural cells at 24 h after reperfusion.Nissl's staining was utilized to observe pathological changes of injured spinal cord segments at 7th day after reperfusion.Re-sults The ROS levels in spinal cord tissue were 49.0 ± 3.5,145.3±11.7,79.0±6.3 and12.8 ± 0.9in SO group,IR group,TP group,and TP + N group at 15 rain after reperfusion,respectively.The ROSlevel in TP group was significantly lower than that in IR group at 15 min after reperfusion (P <0.01),and that in TP + N group was significantly lower than the other three groups (P < 0.01).The p-Akt expression in spinal cord tissue was 0.54 ± 0.06,0.34 ± 0.03,0.42 ± 0.07,and 0.28 ± 0.05 in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The p-Akt expression was sig-nificantly lower in IR group than in SO group (P < 0.01),higher in TP group than in IR group (P <0.05),and lower in TP + N group than in SO group (P <0.01) or in TP group (P <0.01).The percentages of apoptotic cells in spinal cord tissue were (7.5 ± 1.8) %,(31.4 ± 5.5) %,(22.3-± 4.0) %,and (33.3 ± 3.1) % in SO group,IR group,TP group,and TP + N group at 15 min after reperfusion respectively.The percentage of apoptotic cells was significantly higher in IR or TP + N group than in TP group (P <0.01),while there was no significant difference between IR group and TP + N group (P >0.05).The spinal cord sections revealed that pathological changes could hardly be observed in SO group,and mild in TP group but serious in IR and TP + N groups.Conclusion TP produces protective effect on ischemic spinal cord via positive regulation of Akt signaling pathway,and ROS,acting as a signaling molecule,plays an indispensable role in initiating the mechanism.