【摘要】 目的　探讨肝缺血再灌注肝细胞凋亡发生的时空分布、基因表达特点及意义。方法　在观察SD大鼠 (n =4 0 )肝缺血 0 ,3 0 ,4 5 ,60min再灌注 3 ,6,2 4h存活率及病理形态基础上 ,重点观察大鼠 (n =10 0 )全肝血流阻断 3 0min再灌注 0 ,0 5 ,1,3 ,6,12 ,2 4 ,4 8,72 ,96h凋亡细胞分布 (原位末端标记术 ,TUNEL) ,G0 ～G1期细胞、凋亡细胞、增殖细胞指数分布 (流式细胞术 ) ,肝细胞显微及超微结构(光镜、电镜技术 ) ,Fas蛋白、PCNA蛋白 (免疫组化 )及p5 3、bcl- 2基因mRNA表达 (原位杂交 )。结果　缺血 3 0min组多见细胞凋亡 ,细胞应答反应主要为三期 :①急性期 :再灌注 0 .5hFas蛋白首先在血管周围高表达 ;②亚急期 ( 3～ 2 4h) :TUNEL阳性细胞在血管周围高分布 ,并向周围扩展 ;病理形态可见严重变性、细胞皱缩、细胞增殖三种变化 ;G1期细胞数大量减少 ,凋亡峰逐渐升高 ,12h后出现增殖峰 ,相关基因表达增强 ;③恢复早期 ( 2 4～ 96h) :G1期细胞指数回升 ,凋亡峰和增殖峰持续并缓慢下降 ;相关基因表达陆续减弱。结论　肝缺血 3 0min再灌注期机体可能通过下述基因调控途径影响细胞损伤应答反应 :①Fas死亡基因与bcl- 2存活基因的双重调控 ;②细胞凋亡与增殖双重调控 ;③经p5 3基因实现的细胞周期调控。更多还原

【Abstract】 Objective To investigate characteristics of time and areas of liver cell apoptosis and features of gene expression during ischemia/reperfusion and explore their significance. Methods Based on determination of survival rate and pathomorphological changes in 40 SD rats reperfused for 3, 6 and 24 h after hepatic ischemia for 0, 30, 45 and 60 min, we observed pattern of TUNEL cells, indices of G 0-G 1 phase, proliferated and apoptotic cells with flow cytometry, the cell morphological changes with electron microscopy, expression of p53, bcl-2 mRNA with in situ hybridization and Fas protein and PCNA with immunohistochemistry in 100 rats reperfused for 0, 0.5, 1, 3, 6, 12, 24, 48, 72 and 96 h after hepatic ischemia for 30 min. Results Apoptotic phenomenon was mainly found in the group of hepatic ischemia for 30 min. In this group, TUNEL cells appeared near the vascular system at 6 h and developed for all hepatic labor 12 h after the reperfusion. Subsequently, the increase of index of apoptotic hepatocytes was firstly seen 3 h after reperfusion. The expression pattern of Fas was the earliest and strongest. Fas protein and p53 mRNA also expressed firstly near the vascular system between 1 to 3 h and expressed widely from 3 to 72 h. The expression pattern of PCNA appeared from 3 to 96 h. The index of proliferated cells was significantly increased at the 12 h. bcl-2 mRNA also expressed from 3 to 96 h after reperfusion and but it was weaker. Three different morphological characteristics of hepatic cells were observed during the acute (0-3 h after reperfusion), subacute (3-24 h after reperfusion) and earlier repair phase (24-96 h after reperfusion) of liver responses to I/R: severe cell degeneration, cell atrophy and cell proliferation. Conclusion Apoptosis and proliferation are early events after hepatic ischemia/reperfusion and the specific features of this injury. They are probably related to regulating balances of cell death/survival, apoptosis/proliferation and cell cycle transition after hepatic ischemia/reperfusion injury.更多还原