Experimental research on apoptosis induced following microwave coagulation therapy against liver cancer

微波凝血治疗（MCT）是局部热疗的一种，已广泛应用于肝癌患者，临床效果优于PEIT和TAE。尽管有疗效，但治疗后细胞死亡面积扩大的确切机制尚不清楚。本研究的目的是通过对caspase-3活性和DNA片段的连续检测，了解MCT的细胞机制，重点研究热诱导的肝组织凋亡是否发生。采用单极针电极对大鼠肝脏施加1轮MCT，在30 W的条件下，30秒为凝血时间，10秒为解离时间。分别于MCT后2、6、12、24、72、168 h立即处死。测定肝表面面积，评价细胞死亡面积的扩大。通过TUNEL染色和TUNEL阳性细胞计数检测DNA片段的定位和数量，评估细胞凋亡情况。从MCT部位提取的标本中检测caspase-3活性，用于细胞凋亡的酶促评价。caspase-3活性较0 h增加4倍，在MCT后2 h达到峰值。通过TUNEL阳性细胞计数检测，DNA片段在6 h时明显增加，随后减少。肝表面变色面积逐渐增大，持续12 ~ 72 h，此后逐渐消退。MCT显示消融区周围肝细胞发生凋亡。这或许可以解释MCT后细胞死亡面积扩大的机制。

Microwave coagulation therapy (MCT), one of the local hyperthermia, has been widely performed to liver cancer patients, and yielded better clinical results than PEIT and TAE. In spite of the efficacy, the precise mechanism for expansion of cell death area following the therapy has still been unknown. The aim of this study was to understand the cellular mechanism of MCT focusing on whether the heat-induced apoptosis is occurred in the liver tissue by sequential examinations of caspase-3 activity and DNA fragmentation.One bout of MCT was applied to the rat liver using a monopolar needle electrode under setting of 30 W for 30 sec as coagulation time and 10 sec as dissociation time. The rats were sacrificed immediately, 2, 6, 12, 24, 72 and 168 h after MCT. The liver surface area was measured for evaluation of expansion of cell death area. For the assessment of the apoptosis, the TUNEL staining and the TUNEL positive cell counting was examined for the localization and the quantitiveness of DNA fragmentation. The caspase-3 activity in the specimen extracted from MCT site was measured for enzymatic evaluation of apoptosis.The caspase-3 activity showed four-fold increase from the value of 0 h, having a peak at 2 h after MCT. The DNA fragmentation significantly increased at 6 h, and decreased thereafter, assessed by the TUNEL positive cell count. The discolored liver surface area gradually increased until 12 h after MCT, sustained from 12 h to 72 h, and subsided thereafter.It was demonstrated following MCT that the apoptosis of hepatocytes was induced around the ablation area. This may explain the mechanism for the expansion of cell death area subsequent to MCT.