中文摘要：

目的探讨冷冻治疗诱发肿瘤细胞凋亡的原因。方法（1）将GL261胶质瘤细胞（1×10^7个／10μL）注入C57小鼠背部皮下，建立荷瘤小鼠模型，肿瘤直径达15～20mm时将小鼠按随机数字表法分为冷冻治疗组m=17）和假手术组（n=15），前者行冷冻手术治疗，后者只插入冷冻刀但不进行冷冻操作。术后2h提取冷冻肿瘤组织释放物；术后12h、24hTUNEL染色检测肿瘤组织凋亡情况；术后24hWesternblowing实验检测肿瘤组织前体（pro）-Caspase．8、pro．Caspase-8、聚腺苷二磷酸-核糖聚合酶（PARP）的表达。（2）将GL261胶质瘤细胞分为冷冻释放物组、对照组，分别加入1倍浓度的冷冻释放物和等量DMEM，培养12h后TUNEL染色检测细胞凋亡，Westembloaing检测细胞上述凋亡相关蛋白的表达。结果（1）TUNEL染色显示术后12h冷冻治疗组小鼠胶质瘤组织切片S1区呈均一性坏死，S2区出现明显凋亡带（早发凋亡），术后24hS1区呈均一性坏死，S2区凋亡消退，S3区近靶区侧出现一新发凋亡带（迟发凋亡）。Westernblouing检测显示，冷冻治疗组小鼠肿瘤组织S2区pro—Caspase-9和PARP蛋白的表达明显少于S1、S3、S4区，S3区pro—Caspase-8的表达低于S1、S2、S4区，差异均有统计学意SL（P〈0．05）。（2）TUNEL染色显示，与对照组比较，冷冻释放物组GL261胶质瘤细胞凋亡率增加，差异有统计学意义（P〈0．05）。WestembloRing检测显示，与对照组相比，冷冻释放物组细胞pro—Caspase_8、pro—Caspase．9表达下降，差异有统计学意义（P〈0．05）。结论冷冻治疗后肿瘤组织释放物具有诱发胶质瘤细胞凋亡的作用。

英文摘要：

Objective To explore the mechanism of apoptosis after cryotherapy on tumors. Methods （1） GL261 glioma cells （1 ×10^7 cell/10 μL） were injected into the subcutaneous one of C57 mice to establish tumor-bearing mouse models; when the diameter of tumor reached to 15-20 mm, the mice were randomly divided into cryogenic treatment group and sham-operated group （n=10）; mice in the cryogenic treatment group were given surgical cryotherapy, while those in the sham-operated group only performed surgery without cryotherapy. TUNEL was used to detect the cell apoptosis in glioma tissues 12 and 24 h after operation; and Western blotting was employed to detect the protein expressions of pro-caspase-8, pro-caspase-9 and poly-ADP-ribose polymerase （PARP）. （2） GL261 glioma cells were divided into control group and one time cryogenic release group, and DMEM and one time of cryogenic release were given to the two groups, respectively; 12 h after the treatment, TU-NEL was used to observe the cell apoptosis in glioma tissues, and Western blotting was employed to detect the protein expressions. Results （1） TUNEL indicated that the cells in the S1 region of the glioma tissues from mice in the cryogenic treatment group were uniformly died; significant apoptosis was noted in cells of the $2 regionat 12 h after treatment; while, 24 h after treatment, S1 region still showed uniform necrosis, $2 region showed apoptotic regression, and S3 region showed new apoptosis at the target side. Western blotting indicated that pro-caspase-9 and PARP protein expressions at the $2 region were signficantly decreased as compared with those at the S1, S3 and $4 regions （P〈0.05）, and pro-caspase-8 protein expression at the S3 region were signficantly reduced as compared with those at the S1, S2 and S4 regions in the cryogenic treatment group （P〈0.05）. （2） TUNEL showed that significantly increased GL261 glioma cell apoptosis rate was noted in the one time cryogenic release group as compared with that in the control gro