低剂量FK506预处理诱导移植物对缺血再灌注损伤耐受的影响

Pharmacological preconditioning with low-dose FK506 reduces subsequent ischemia-reperfusion injury in rabbit allograft knee joint

目的观察并初步探讨低剂量FK506预处理诱导带血供同种异体膝关节移植时移植物对缺血再灌注损伤的耐受及其可能的机制。方法用24只日本大耳白兔,建立带血供同种异体膝关节移植异位动物模型,并将其分为IR组(缺血再灌注组)、FK506组(低剂量FK506预处理组)及ST组(对照组),缺血1h后再灌注,然后取缺血再灌注前及制备再灌注后12h的相同组织(以肌肉组织为例)进行苏本精-伊红染色、透射电镜、流式细胞仪、RT-PCR、WesternBlot、DNA梯度电泳及SOD、NO与MDA的分光光度计检测。结果结果发现,HE染色显永,IR组组织结构紊乱,细胞疏松;而FK506组中组织结构清晰、细胞完整。TEM显示IR组线粒体肿胀,空泡化;FK506组线粒体完整,无明显空泡化。SOD、NO及MDA检测显示:IR组中NO及MDA含量明显增加,SOD活性下降,而FK506绀中NO、MDA含量增加不明显,SOD活性较强。DNA梯度电泳显示,FK506预处理后DNA梯度较IR组减弱。流式细胞仪检测显示:低剂量FK506预处理后,细胞的凋亡率可从20%降至10%左右,而Fas及FasL的表达则明显降低。RT-PCR检测显示,经FK506预处理后,TNF及IL-1mRNA水平的表达减弱,而与此同时,HSP70在mRNA水平的表达则明显增强,WesternBlot检测仪显示HSP70在相应时间蛋白水平亦是明显增强的。结论低剂量的FK506(0.3mg/kg)预处理在带血供同种异体膝关节移植时可以诱导移植物对缺血再灌注损伤的耐受。

Objective To explore the effect of pharmacological preconditioning wtith low-dose FK506 reducing subsequent ischemia-reperfusion injury in rabbit knee joint and to explore the possible mechanism ofthat. Methods Twenty four animal model of allograft heterootopic knee joint were established and divided randonly into three groups： IR group （lschemia-reperfusion group）, FK506 group （FK506 preconditioning group） and ST group（ Sham control group）. Vessels were clamped for 1 hour, and then grafts received reperfusion. Tissues（for example muscles tissues） of pro-ischemia and reperfusion after 12 hours were obtained,and they were observed by microscope and electron microscope and were analysised with reverse transcription polymerase chain reaction（RT-PCR）, Western Blot, DNA ladder, flow cytometer. Moreover, the contents of MDA, NO and SOD in these tissues were examined by spectrophotometer. Results As a result, we observed disoranized tissues and swollen cells in IR groups but trenchant tissues and intact cells in FK506 groups by microscope, vacuolated and tumid mitochondrion in IR groups but non-vacuolated and intact that in FKS06groups. With FK506 preconidtioning, the contents MDA and NO were distinctly reduced but significant raisedin FK506 group. Compared with IR group, DNA ladder of FK506 group were weakly, and apoptotic ractes reduce from 20% to 10% and the expression of TNF, IL-1, Fas and FasL mRNA obviously down-regulated. At the same time, expression of HSP70 significantly up-regulated on protein and mRNA levels with RT-PCR andWestern blotting analysis in FK506 group. Conclusion Pharmacological preconditioning with low-dose FK506 （0. 3mg/kg） can induce tolerance of ischemia-reperfusion injury in rabbit allograft knee joint. Over-expression of HSP70 may be key factor of that.