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中华普通外科学文献(电子版) ›› 2016, Vol. 10 ›› Issue (01) : 24 -30. doi: 10.3877/cma.j.issn.1674-0793.2016.01.003

所属专题: 文献

论著

兔肌肉移植瘤纳米刀、冷冻、射频消融的对比实验研究
宁周雨1, 王鹏1, 陈颢1, 解婧1, 林钧华1, 朱晓燕1, 徐立涛1, 陈其文1, 高嵩1, 宋利斌1, 姜峰2, 孟志强1,()   
  1. 1. 200032 上海,复旦大学附属肿瘤医院综合肿瘤科
    2. 262200 潍坊诸城中医医院肿瘤科
  • 收稿日期:2015-10-20 出版日期:2016-02-01
  • 通信作者: 孟志强

Comparative study of Nano knife, cryoablation, radiofrequency ablation in the muscle xenografts in rabbits

Zhouyu Ning1, Peng Wang1, Hao Chen1, Jing Xie1, Junhua Lin1, Xiaoyan Zhu1, Litao Xu1, Qiwen Chen1, Song Gao1, Libin Song1, Feng Jiang2, Zhiqiang Meng1,()   

  1. 1. Department of Integrative Cancer, Fu-dan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan Uni-versity, Shanghai 200032, China
    2. Department of Oncology, Weifang Zhucheng Chinese Medicine Hospital, Weifang 262200, China
  • Received:2015-10-20 Published:2016-02-01
  • Corresponding author: Zhiqiang Meng
  • About author:
    Corresponding author: Meng Zhiqiang, Email:
引用本文:

宁周雨, 王鹏, 陈颢, 解婧, 林钧华, 朱晓燕, 徐立涛, 陈其文, 高嵩, 宋利斌, 姜峰, 孟志强. 兔肌肉移植瘤纳米刀、冷冻、射频消融的对比实验研究[J/OL]. 中华普通外科学文献(电子版), 2016, 10(01): 24-30.

Zhouyu Ning, Peng Wang, Hao Chen, Jing Xie, Junhua Lin, Xiaoyan Zhu, Litao Xu, Qiwen Chen, Song Gao, Libin Song, Feng Jiang, Zhiqiang Meng. Comparative study of Nano knife, cryoablation, radiofrequency ablation in the muscle xenografts in rabbits[J/OL]. Chinese Archives of General Surgery(Electronic Edition), 2016, 10(01): 24-30.

目的

探讨肌肉移植瘤纳米刀(IRE)、冷冻、射频消融术后的肿瘤凋亡、机体免疫及血管生成因子情况,观察不同微创技术对肌肉移植瘤的抗肿瘤效果,为临床应用提供一定的实验依据。

方法

兔肌肉移植瘤模型8只,并随机分为正常对照组、纳米刀治疗组、冷冻治疗组及射频治疗组,每组2只。动态观察治疗过程中新西兰大白兔的生命体征以及治疗后并发症,术后24 h全部处死,分离血浆及血清检测,取瘤块包埋切片,HE染色,免疫组织化学方法检测Bcl-2、HSP70和VEGF在肌肉移植瘤中的表达,并运用Tunel法检测细胞凋亡。

结果

肉眼观肿瘤表面血管丰富,触之质地软;各组术后治疗区域可见明显凝固性坏死改变,冷冻及射频术后临近肌肉组织充血水肿明显。HE染色显示纳米刀组治疗区域边界清晰,冷冻及射频术区仍可见部分存活细胞。Tunel结果显示纳米刀治疗组可以明显增加肿瘤细胞凋亡,各治疗组均可通过升高血浆Caspase-3水平及降低术区Bcl-2表达促进肿瘤组织凋亡,升高血浆TNF-α水平激发机体抗肿瘤免疫,同时促使血浆VEGF因子分泌短期内升高;肿瘤组织术区边缘HSP70及VEGF因子表达水平各组不一。术后血清学检测结果显示各组治疗后新西兰大白兔的肌酸激酶水平升高,CTnI及肝肾功能未见异常。

结论

纳米刀、氩氦刀冷冻、射频均能够通过直接消融、诱导细胞凋亡、产生特异的抗肿瘤免疫效应来抑制兔肌肉移植瘤的生长,且治疗方法安全、有效。纳米刀在诱导细胞凋亡、周围重要脏器的保护及治疗时间短均有明显优势,为其进一步的临床应用提供了动物实验依据。

Objective

To investigate the apoptosis, immunity and angiogenesis factor changes of rabbits after irreversible electroporation (IRE), cryoablation, radiofrequency ablation (RFA) of muscle can-cer xenografts, and to observe the antitumor effect and security of diversiform minimally invasive tech-niques for muscle cancer xenografts.

Methods

Eight muscle cancer xenograft models were randomly di-vided into IRE, cryoablation, RFA and control group, each group contained 2 rabbits. All the rabbits were executed after 24 hours. Liver, kidney function and creatinekinase were tested. ELISA methods were used to detect CTnI, Caspase-3, TNF-α and VEGF levels in the plasma. Tunel assay was used to detect the apoptosis of tumor tissue. Immunohistochemistry was applied to detect Bcl-2, HSP70 and VEGF expres-sion in muscle cancer xenografts.

Results

Rich blood vessels were showed on the tumor surface, with soft texture. Obvious coagulation necrosis and edema were found in the adjacent muscle tissue. HE stain-ing showed a clear borderline between the necrotic and normal area in the IRE group, visible liver cells in the cryoablation and RFA group. Tunel assay showed that IRE can significantly increase the apoptosis in muscle cancer cells compared with other groups. Each group could promote tumor apoptosis by increasing plasma Caspase-3 level and reducing Bcl-2 expression in the treatment area, inspired the antitumor im-munity by elevating plasma TNF-α level and induced to the elevation of plasma VEGF secretion in the short time after treatment, HSP70 and VEGF factor expression were variant in edge of the treatment area. Post-treatment serological test showed that liver, kidney functions and CTnI were within the normal range, except for significant higher levels of creatine kinase.

Conclusions

IRE, cryoablation, and RFA can in-hibit the growth of liver tumors via ablating directly, inducing apoptosis, producing specific anti-tumor im-mune response, it is safe and effective. IRE has advantages in the induction of apoptosis, protection of im-portant viscera nearby and short ablation time. This study may support further clinical application of IRE on human beings with the evidence from animals.

图1 A:实验兔肌肉移植瘤大体标本,肿瘤表面可见丰富的新生血管,局部凸起,边界不清。B-D:纳米刀、冷冻、射频治疗实验兔肌肉移植瘤示意图。E:对照组肿瘤细胞密集,不规则排列紧密,间质少,其大小和形态各异,边缘肌肉组织内可见浸润性癌结节(HE,×400)。F:纳米刀组术区癌细胞核固缩、碎裂。肿瘤血管内血栓形成,内见红细胞碎裂物沉积,治疗区内血管腔结构完整(HE,×400)。G:冷冻组术区边缘不规则,治疗区红染,可见坏死吸收区,内部分肿瘤细胞仍见存活(HE,×400)。H:射频组术区红染,消融区边缘不规则,散在的存活肿瘤细胞,周围可见大量炎症细胞浸润及凋亡的肿瘤细胞(HE,×400)
图2 A实验兔各组术后血浆Caspase-3水平均升高,冷冻组水平最高;血浆TNF-α水平,其中射频组TNF-α水平升高最显著;血浆VEGF水平均有所升高,冷冻组为著。(ns为差异无统计学意义;0.01<**P<0.05;*** P<0.01;A为正常对照组,B为纳米刀组,C为冷冻组,D为射频组)
图3 不同术式治疗后边缘细胞凋亡(Tunel ×200),凋亡率各组均升高,纳米刀组升高最为显著(A-D分别为正常对照组、纳米刀组、冷冻组,射频组)
图4 A, D:Bcl-2表达于胞质,部分胞核亦见表达(Envision法×200);射频组术后术区边缘Bcl-2表达明显下降最为显著。B, E:纳米刀术后术区边缘部分肿瘤组织及正常肌肉组织可见多发HSP70染色阳性细胞,主要分布于肌束周围周围结缔组织(Envision法×200);各组术后术区边缘HSP70水平表达不一,以射频组升高显著,纳米刀组无升高,而冷冻组出现降低。CF:VEGF表达于胞质,术区细胞破裂,部分定位不清,周围肌肉组织少量表达(Envision法×200);术后治疗区边缘VEGF表达不一,于冷冻组下降显著,纳米刀组较正常对照组差异无统计学意义,而射频组VEGF水平表达明显较术前升高。(ns为差异无统计学意义;*P<0.05;0.01<**P<0.05;***P<0.01,A为正常对照组,B为纳米刀组,C为冷冻组,D为射频组)
表1 新西兰大白兔不同术式术后的肝肾功能、淀粉酶、肌酸激酶及肌钙蛋白I变化
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