氩氦刀冷冻后肝细胞中线粒体变化的电镜观察

Ultrastructurally Pathological Changes of Mitochondria in Hepatocytes of Rabbit Liver Frozen by Cryocare

目的探讨氩氦刀冷冻后不同时间点兔肝细胞中线粒体的形态变化及其生物学反应,为临床上肝脏肿瘤氩氦刀治疗提供实验依据。方法动物分冷冻5、10min两组,均用直径2mm氩氦刀置于兔肝一叶表面,冷冻后分别在一系列时间点上取冷冻处肝组织,按透射电镜制样技术制样,在电镜下观察各组肝脏的肝细胞中线粒体在各时间点上的超微结构变化。结果冷冻后0.5天时,肝细胞中线粒体肿胀,部分外膜破裂,基质中密度均质变化,含染色质物质聚集成大小不等形状不同的团块状分布于边部。4天时,线粒体基质溶解,外膜破裂明显。8天时,中心区线粒体轮廓消失,周边区尚存含染色质物团块堆成的轮廓。16天时,被结缔组织包围的肝组织残余中尚存少量含染色质物质团块。30天时,冷冻区基本上被结缔组织充填,其内肝组织残余几乎消失。结论在氩氦刀冷冻5、10min后,肝细胞中线粒体随时间延长其外膜、基质及染色质发生一系列的质变过程,最终是溶解消失。在此过程中,线粒体死亡是一个不可逆的过程,是判定细胞死亡及临床上确定氩氦刀冷冻时间的根据之一。

Objective To explore the ultrastructurally pathological changes of the mitochondria in rabbit liver cells frozen by CryocareTM and provide a laboratory evidence for clinical liver cancer treatment with cryocare. Methods A 2 mm diameter probe was set on the surface of rabbit liver for 5 or 10 min. The hepatic tissues were taken respectively at 0. 5, 1, 2, 4, 8, 16, 30 days after frozen and observed under transmission electron microscope. Results At 12th hours after the liver was frozen, the mitochondria swelled, the outer membrane of some cells disruptted, the stroma became homogenated in moderate density and the chromatin of mitochondria concentrated into irregular blocks of different sizes mostly located at out board edge. On the 4th day, the blocks containing mitochondrial chromatin dispersed in the cytoplasmic remnant and stroma dissolved or disappeared. On the 8th day, the mitochondria completely disappeared at central area of the frozen tissues. On the 16th day, the liver cell morph could not be identified, only some cellular remnant could be seen in central area. On the 30th day, the frozen area nearly disappeared, and was substituted with some connective tissues. Conclusion After 5, 10min freezing, the mitochondria in liver cells underwent swelling, disruption and their chromatin accumulated and finally dissolved. It is deemed that the death of liver tissues after cryocare freezing is a natural and irreversible process.