移植瘤株冷冻保护的实验研究

Experiment of preserving efficiency of xenografts freezed in liquid nitrogen

本文介绍人肝癌细胞株细胞及其移植瘤组织经冻存、复苏后检测琥珀酸脱氢酶(SDE)活性、成瘤率和电镜检查。选择10％DMSO或10％甘油作为冷冻保护剂来冻存细胞。它们先在冰箱里缓慢降温冷却（-0．8℃／min），后在液氮罐口的液氮气中冷冻（-10℃／min）1天。最后置入液氮内。冷冻保护后的冻存或组织复苏后显示高的SDE活性，10％DMSO冻存的细胞SDE活性比10％甘油者高，两者相比有显著差别（P＜0．01）。尽管两组复苏后的接种成瘤率相同（80％），但它们移植25天后，前者移植瘤瘤重比后者重1．66倍。电镜下，两种冻存液冻存的细胞都有“暗”和“明”细胞，它们都有完整的线粒体膜和质膜。推荐10％DMSO作为移植瘤冷冻保护液。

It has not been done into detail that whether human Xenografted tumors could be frozen in liquid nitrogen(- 196 ),thawed and recovered. A human hepatocellular carcinoma cell line (H91O1 ) and its xenografts were tested by succino dehydrogenase (SDE)activity (MTT test) after freezing and thawing and being studied by electron microscope.Two preservative liquids included lO% DMSo or 1o% glyceroI were adopted for the freezing in which the cells or tissues were controlled to be reduced to a minimum rate of cooling (-0.8t / min) in the gas of liquid nitrogen for 24 hours and programmed in liquid nitrogen.It was shown that H9lOl cells or tissues after freezing and thawing presented a high SDE activity (72%~ 93%). The difference of SDE activity between the cells or tissues preserved in 10% DMSO and that preserved in lO% blycerol were significant (P< 0.01 ). The inoculating efficiency of xenografts stored in 10% DMSO was same to that stored in lO% glycerol. Observation by electron microscoPe, tumor cells frozen by both of the preservative medium showed more'black'cell and less'light'cells. Both'black'and'light'ce1ls showed an integral structure of mitochondria membrane and cytoplasmic membrane. Although there was not a general acceptance of the claims that DMSO was preferable to glycerol as an agent for pre venting different organism from freezing damages, it was satisfactory that xenografts pre served in 10% DMSO could improve the survival rates of tumor cells frozen in liquid nitrogen