冷冻消融后残存肿瘤与上皮-间质转化关系的实验研究

目的 探究不完全冷冻消融对前列腺癌RM-1细胞生物学行为的影响及其产生机制.方法 前列腺癌RM-1细胞放入-20℃冰箱中冷冻5 min,37℃水域复温,待细胞状态恢复后重复冷冻10 min、15 min.培养ld后镜下观察细胞形态.20只C57/BL小鼠构建荷瘤模型,随机分为对照组与不完全冷冻消融组.不同时间点测量肿瘤大小,14d时处死小鼠、取肺组织行HE染色,统计肿瘤肺转移枚数.Transwell法检测细胞迁移及侵袭能力变化,免疫印迹检测相关蛋白表达水平,酶联免疫吸附试验(ELISA)法检测细胞上清液转化生长因子(TGF)-β分泌量.结果 经不完全冷冻消融的RM-1细胞排列紊乱,形态发生改变,可有触角结构形成.术后3、7d不完全冷冻消融组肿瘤体积略小于对照组,但仅术后7d差异有统计学意义(P=0.019),术后10、14 d肿瘤体积基本相等.不完全冷冻消融组肿瘤肺转移枚数明显多于对照组(P＜0.001).Transwell试验显示不完全冷冻消融组细胞迁移及侵袭能力强于对照组(P＜0.05).免疫印迹检测显示,不完全冷冻消融组与对照组相比,N-cadherin、MMP-9、vimentin、表达上调,E-cadherin表达下调.ELISA检测结果显示,不完全冷冻消融组细胞上清液中TGF-β分泌增多.结论 不完全冷冻消融可使RM-1细胞迁移及侵袭能力增强,增加荷瘤小鼠肿瘤肺转移枚数,影响上皮-间质转化相关蛋白表达.

The relationship between the remnant tumor and the epithelial-mesenchymal transition after cryoablation:an experimental study

Objective To investigate the effect of incomplete cryoablation on the biological behavior of prostatic cancer RM-1 cells and its mechanism.Methods RM-1 cells of prostatic cancer were placed in -20℃ icebox to be frozen for 5 min.After the recovery of the cell state,the RM-1 cells were frozen again for 10 min and 15 min successively.After culture for one day,the cellular morphology was microscopically examined.A total of 20 C57/BL mice were used to establish the tumor-bearing models,which were randomly and equally divided into the control group and the incomplete cryoablation group with 10 mice in each group.At scheduled time points the tumor lesion size was measured for all mice.The mice were sacrificed at 14 days,the lung tissues were collected and were stained with lE;the numbers of metastatic lesions in the lung were calculated.Transwell assay was used to test the cell migration and invasion,immuno-blotting method was adopted to determine the epithelial-mesenchymal transition-related (EMT-related) protein expression level,and the enzyme-linked immunosorbent assay (ELISA) was employed to check the secretion volume of transforming growth factor-beta (TGF-β).Results After incomplete cryoablation,RM-1 cells became disorderly arranged,their morphology was changed,and antenna structure might be formed.At 3 and 7 days after cryoablation,the tumor size in the incomplete cryoablation group was slightly smaller than that in the control group,but only the difference at 7 days after cryoablation was statistically significant between the two groups (P=0.019).At 10 and 14 days after cryoablation,the tumor volume of the two groups was almost equal.The pulmonary metastatic lesions in the incomplete cryoablation group were obviously much more than those in the control group (P＜0.001).Transwell assay indicated that the cell migration and invasion ability in the incomplete cryoablation group was stronger than that in the control group (P＜0.05).Immuno-blotting test revealed that,when compared with the control group,in the incomplete cryoablation group the expressions of N-cadherin,MMP-9 and Vimentin were up-regulated,while the expression of E-cadherin was downregulated.ELISA test showed that increased secretion of TGF-β was observed in the incomplete cryoablation group.Conclusion Incomplete cryoablation can enhance the migration and invasion ability of RM-1 cells,increase the number of pulmonary metastatic lesions in tumor-bearing mice,and affect the EMT-related protein expression level.