不可逆性电穿孔致山羊肝脏组织凋亡与坏死的实验研究

Experimental study on apoptosis and necrosis of goat hepatocytes induced by irreversible electroporation

目的评价不可逆性电穿孔(IRE)消融山羊肝脏组织的效果,以及消融区域肝细胞坏死、凋亡与电场分布的关系。方法取4～6月龄山羊作为研究对象,对其给予IRE处理:经彩色超声探头引导电极针经皮定位肝脏,施加120个脉冲电场(固定电压2000V、脉宽100μs、频率1Hz)。24h后处死动物,取电极针周围肝脏组织标本(包括脉冲电场处理区域中心部分和处理区域与正常组织交界区)和正常组织(作为对照)。将标本进行处理后,分别行HE染色观察组织的病理学变化;采用DNA ladder和TUNEL法检测组织细胞凋亡,采用流式细胞仪分析细胞周期。结果病理学观察可见,消融处理区的肝细胞完全坏死,与正常组织分界清晰,围绕电极针的中心区域呈现凝固性坏死。TUNEL法显示凋亡细胞主要集中在处理区与正常组织的交界区。DNA ladder法在处理区与正常组织交界区组织检测到400～1000bp的梯状条带。流式细胞术分析显示,脉冲电场处理区细胞DNA二倍体峰前面出现亚二倍体凋亡峰。结论采用IRE消融山羊肝脏组织,细胞在电场强度分布最高处(即围绕电极针的中心区域)呈现凝固性坏死;在电极针周边处理区域,随电场强度衰减,肝细胞仍呈现出凋亡迹象。

Objective To investigate the ablation effects of irreversible electroporation（IRE） on goat liver tissues,and analyze the relationship of apoptosis and necrosis of hepatocytes to the distribution of electric fields.Methods IRE was performed on goats of 4-6 months old as follows： the electrodes were targeted to goats’ liver tissue under ultrasonic guidance,and the pulse electric fields（PEFs） were exerted with 120 pulses of permanent duration（100μs）,frequency（1Hz） and voltage（2000V）.Animals were sacrificed 24 hours after treatment,the samples of liver tissues were harvested,including the tissues surrounding the electrodes and that of the border area,with normal tissue as control.The specimens were prepared for HE staining to observe the histopathological changes.DNA ladder analysis and TUNEL test were performed to determine apoptosis,and flow cytometry（FCM） was employed to analyze the changes in cell cycle.Results Pathologically,cell necrosis was found in ablation zone with a distinguish border line between the normal tissue,and coagulate necrosis was seen in the area surrounding electrodes.TUNEL test showed apoptotic cells,with heavy black staining of the nuclei and light brownish yellow in cytoplasm,and they were mainly aggregated in the border between treated and normal tissues.In this area the ladder strips of 400 to 1000bp were also detected by DNA electrophoragram.Moreover,hypodiploid peak was found,prior to the diploid peak,by FCM analysis in the hepatocytes obtained from ablated zone.Conclusion In goats’ liver tissue ablated by means of IRE,coagulation necrosis may appear in the areas under highest electric field intensity surrounding the central area of electrodes,and the apoptosis of hepatocytes may be found in the border areas along with the attenuation of electric field intensity.