【作者】 张克勤；

【导师】 张积仁；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2007， 博士

【摘要】 研究背景肝癌是我国发病率第三位的恶性肿瘤，是最常见的恶性肿瘤之一。早期肝癌最有效的治疗方法是手术切除。但由于其发病隐匿、就诊较晚及合并肝硬化致肝功能差等原因，手术切除率仅20％～30％。对不能手术切除的中晚期肝癌患者采用微创治疗，仍可取得较好疗效。随着现代物理学技术向医学领域的延伸以及现代影像技术、微电子学、计算机信息处理等在科学领域的应用，微创治疗已成为中晚期肝癌治疗中不可缺少的重要组成部分。肝癌常用的微创治疗可以分成两大类：一类是血管性微创治疗，包括肝动脉灌注化疗（TAI）、肝动脉栓塞（TAE）和肝动脉栓塞化疗（TACE）；另一类是非血管性微创治疗，包括经皮化学消融和经皮物理消融，前者常用的有经皮无水乙醇注射（PEI）、经皮穿刺乙酸注射（PAI），后者包括氩氦刀冷冻消融、射频消融（RFA）、微波凝固（MCT）、激光间质热疗及高强度聚焦超声等。其中氩氦刀冷冻消融、射频消融（RFA）和微波凝固（MCT）在临床上被广泛应用。这三种微创治疗都属于经皮物理消融技术，即采用冷或热效应来杀灭肿瘤细胞，达到“一次性损毁”的治疗效果。所谓“一次性损毁”是指使用该项技术治疗肝癌时能一次性毁灭、杀死其治疗范围内的肿瘤细胞。因此，在使用这三种微创技术治疗肝癌时，其临床疗效具有较强的可比性。上述三种微创治疗技术为目前最常用的中晚期肝癌治疗方法，各有其优缺点，临床疗效各有千秋，单独或联合其它微创技术用于治疗中晚期肝癌的临床疗效和实验研究报道较多。然而这三种微创治疗技术相比较究竟那一种方法临床疗效更优?消融靶区评价如何?目前国内外尚无相关实验对比和临床对比研究的报道。兔VX₂肝癌模型是少数建立在大动物身上的肝癌模型，比较适合于肝癌的微创治疗实验研究。其瘤源VX₂肿瘤细胞株起源于Shope病毒诱发的兔乳头状瘤衍生的鳞癌，经过72次移植传代后正式建立株，命名为VX₂。是目前最为理想的肝癌实验模型之一，被广泛应用于肝癌肿瘤学、肿瘤治疗学、放射生物学、抗癌药物药代动力学及肿瘤影像学等实验研究。本实验研究的目的在于对兔VX₂肝癌模型使用上述三种微创治疗技术进行分组治疗，通过观察肿瘤消融靶区大小情况、肿瘤细胞坏死及残留情况、肿瘤转移情况、治疗前后免疫功能变化以及兔的生存期来比较各自的治疗效果，探讨这三种微创治疗技术的作用机制及临床疗效，为临床上正确地选择这三种微创技术治疗肝癌提供理论依据和实验依据。第一章兔VX₂肝癌模型的制作目的事实证明，微创治疗已成为中晚期肝癌综合治疗中不可缺少的重要组成部分，是肝癌非手术疗法中的首选方法。但目前仍然存在许多需要研究的问题，如进一步完善肝癌供血理论、微创治疗的远期疗效和复发、微创治疗与细胞凋亡及肿瘤耐药的关系等。另外各种非血管性的微创治疗如冷冻、射频、微波等仍需要继续研究。这就需要建立一个稳定的较大的动物模型。理想的肝癌模型应与人类的肝癌生物学行为相同或相近，造模成功率高而动物死亡率低；研究中可重复性好，造模方法简便易行；动物大小、体形等需适合于所做的病因、诊断、治疗的实验研究；所用方法对人体无害或较少危害，环境污染小。以往肿瘤模型大多建立在鼠身上，但由于鼠身体较小，血管细，所形成的肿瘤亦小，不适合作为肿瘤的微创治疗研究。其它动物如猪、犬则很少被采用，因为这两种动物的诱生型肝癌不同，个体间可比性差，移植型肝癌则因排斥成功率低，难以成活。而兔体型较大，比较适合于肝癌的微创治疗实验研究。兔VX₂肝癌模型是少数建立在大动物身上的肝癌模型。其瘤源VX₂肿瘤细胞株起源于Shope病毒诱发的兔乳头状瘤衍生的鳞癌，经过72次移植传代后正式建立株，命名为VX₂。移植性兔VX₂肝癌模型具有许多优点：1、为目前国内最大动物的实验性肝癌模型；2、实验证明该肿瘤为富血管性肿瘤，其供血动脉主要为肝动脉，类似于人类原发性肝癌；3、病理学上肿瘤为巨块型实体瘤、侵润性生长，血供丰富，类似于巨块型肝癌；4、模型制作简便易行，价格相对较低廉，且实验周期短，一般兔VX₂肝癌生长至直径3cm大小仅需3—4周。因此兔VX₂肝癌已成为目前最为理想的肝癌实验模型之一，被广泛应用于肝癌肿瘤学、肿瘤治疗学、放射生物学、抗癌药物药代动力学及肿瘤影像学等实验研究。本实验采用开腹穿刺接种法制作兔VX₂肝癌模型。方法兔VX₂肝癌模型常选择兔肝左叶作为细胞株种植地，种植方法有1．开腹包埋接种；2．开腹穿刺接种；3．经皮直接穿刺接种；4．癌细胞悬液接种。本实验取试验用新西兰大白兔80只，采用开腹穿刺接种法制作兔VX₂肝癌。尽可能保证接种的部位、深度相一致，利于增加实验的可比性。该方法较简单、经济、成功率高，开腹直视下接种部位准确，种植2—3周，肿瘤可长至1—3cm，利于微创靶向消融技术的实施。结果80只新西兰大白兔中有76只经CT检查和病理检查证实接种成功，接种成功率达95％，与文献报道的结果基本一致。结论采用开腹穿刺接种法制作兔VX₂肝癌模型简单、经济、成功率高，开腹直视下接种部位准确，种植2—3周，肿瘤可长至1—3cm，是进行肝癌微创治疗实验研究较理想的肝癌模型。第二章氩氦刀冷冻消融和射频、微波热凝固对兔VX₂肝癌消融作用的对比研究目的临床上常用的微创治疗可以分成两大类：一类是血管性微创治疗，包括肝动脉灌注化疗（TAI）、肝动脉栓塞（TAE）和肝动脉栓塞化疗（TACE）；另一类是非血管性微创治疗，包括经皮化学消融和经皮物理消融，前者常用的有经皮无水乙醇注射（PEI）、经皮穿刺乙酸注射，后者包括氩氦刀冷冻、射频消融（RFA）、微波凝固（MCT）、激光间质热疗及高强度聚焦超声等。在肝癌的微创治疗中，氩氦刀冷冻、射频消融及微波凝固是三种常用的物理消融方法，它们都是通过冷或热效应来杀灭肿瘤细胞。氩氦刀冷冻治疗肿瘤的主要生物学效应是由氩气产生的冷冻完成。但氦气产生的热效应亦有一定的作用，因为短时间冷冻和升温过程会造成细胞内外冰晶爆裂，对肿瘤细胞的损毁更为彻底。超低温冷冻引起癌细胞死亡的作用机制：1．细胞脱水和皱缩；2．细胞电解质毒性浓缩和PH值改变；3．细胞膜脂蛋白成分变性；4．细胞内冰晶形成和冰晶的机械性损伤；5．血流淤滞和微血栓形成；6、免疫效应。射频消融（RFA）和微波凝固（MCT）属于热消融的范畴，都是通过高温来杀死癌细胞，高温引起癌细胞死亡的作用机制：1．肿瘤组织淤血、缺氧；2．肿瘤组织内PH降低，酸性增多，溶酶体增多，溶酶体酶性化；3．瘤细胞DNA、RNA及蛋白合成受到抑制；4．机体免疫反应增强。在使用这三种物理消融微创技术治疗肝癌的过程中，它们所产生的消融靶区大小和对靶区内肿瘤细胞的杀灭能力与其治疗效果成正比。本实验的目的是采用氩氦刀冷冻、射频消融及微波凝固对兔VX₂肝癌进行消融，然后通过对标本的大体观察和病理检查，了解肿瘤消融靶区大小情况、肿瘤细胞坏死及残留情况并加以比较，探讨这三种微创技术对兔VX₂肝癌的消融作用机制。方法取制作成功VX₂肝癌模型的新西兰大白兔（接种VX₂细胞株后3周）27只，采用螺旋CT平扫检查测量肿瘤的纵经和横经后随机分为三组，每组9只，即氩氦刀冷冻组（A组）、射频消融组（B组）和微波凝固组（C组）。将实验兔用3％戊巴比妥钠（1ml／kg）全麻后固定于兔手术台上。在上腹部正中原伤口疤痕处作2—3cm长切口打开腹腔，暴露肝脏，用镊子轻夹肝左叶将其拉出体外，观察并估计肿瘤的大小。然后A、B、C组分别采用美国Endocare公司生产的氩氦刀冷冻系统、珠海市和佳医疗设备有限公司生产的HGCF—3000冷极射频肿瘤治疗机和南京庆海微波电子研究所生产的MTC—3C组织间肿瘤微波凝固治疗仪进行分组消融。分别将氩氦刀针、射频电极针（单极冷循环）和微波电极针沿肿瘤长轴（纵经）直接穿刺肿块中心，针尖达肿瘤对侧边缘，然后进行消融。各组消融时间均为该治疗设备用于人肝癌临床治疗时间的1／3（分别为5分钟、5分钟和3分钟）。3天后处死全部兔子，切下肿瘤标本，福尔马林溶液固定，送作病理检查。应用SPSS软件进行统计学分析。计量资料用均值±标准差。统计前经方差齐性检验；满足齐性，三组间比较用单向方差分析，两两比较用S-N-K法；若不满足齐性用Welch方法，两两比较用Games-Howell法。P＜0.05有统计学意义。结果1．消融靶区横径：A组（氩氦刀冷冻组）为2.28±0.12cm、B组（RFA组）为1.96±0.09cm、C组（MCT组）为1.93±0.10cm，三组比较差异有显著性（F=31.138，P=0.000）；A组与B组比较、A组与C组比较差异有显著性（P=0.000），B组与C组比较差异无显著性（P=0.653）。2．消融靶区平均面积：A组为5.79±1.34cm²、B组为4.40±0.43cm²、C组为4.31±0.59cm²，三组比较差异有显著性（F=8.047，P=0.002）；A组与B组比较、A组与C组比较差异有显著性（P=0.003，P=0.002），B组与C组比较差异无显著性（P=0.836）。3．消融靶区肿瘤完全消融率（消融靶区完全覆盖整个肿瘤区）：A组为88.9％（8只兔，占8／9）、B组为44.44％（4只兔，占4／9）、C组为33.33％（3只兔，占3／9）。三组比较差异有显著性（x²=6.300，P=0.043）；A组与B、c组比较差异显著（P＜0.05），B、C组之间比较差异不显著（P＞0.05）。4．消融靶区肿瘤细胞残留率：A组为22.22％，B、C组均为77.78％，三组比较差异有显著性（x²=7.670，P=0.022）；A组与B、C组比较差异显著（P＜0.05），B、C组之间比较差异不显著（P=1.000）。5．消融靶区肿瘤细胞完全坏死率：A组为66.67％，高于B组和C组（50.00％）。而B组和C组均为50.00％，差异无显著性。结论三种微创治疗在消融兔VX₂肝癌中：无论是在消融靶区面积和横径方面、消融靶区肿瘤完全消融率方面，还是在消融靶区肿瘤细胞残留率方面和消融靶区中肿瘤细胞完全坏死率方面，氩氦刀冷冻均优于RFA和MCT；而RFA和MCT效果相当。提示氩氦刀冷冻在治疗兔VX₂肝癌中其临床疗效可能优于RFA和MCT。第三章氩氦刀冷冻消融和射频、微波热凝固对兔VX₂肝癌治疗作用的对比研究目的肝癌是我国发病率第三位的恶性肿瘤，是最常见的恶性肿瘤之一。早期肝癌最有效的治疗方法是手术切除。但由于其发病隐匿、就诊较晚及合并肝硬化致肝功能差等原因，手术切除率仅20％～30％。对不能手术切除的中晚期肝癌患者采用微创治疗，仍可取得较好疗效。因此，微创治疗已成为中晚期肝癌治疗中不可缺少的重要组成部分。其中氩氦刀冷冻、射频消融（RFA）和微波凝固（MCT）在临床上被广泛应用。这三种微创治疗都属于经皮物理消融技术，即采用冷或热效应来杀灭肿瘤细胞，达到“一次性损毁”的治疗效果。所谓“一次性损毁”是指该项技术治疗肝癌时能一次性毁灭、杀死其治疗范围内的肿瘤细胞。因此，在使用这三种微创技术治疗肝癌时，其临床疗效具有较强的可比性。然而这三种微创治疗技术当中究竟那一种方法临床疗效更优?目前国内外尚无相关实验对比和临床对比研究的报道。本实验研究的目的在于对兔VX₂肝癌使用氩氦刀冷冻、射频消融及微波凝固三种微创治疗技术进行分组治疗，并与手术切除组和对照组比较，通过观察肿瘤残留和转移情况、治疗前后免疫功能变化以及兔的生存期来比较各自的治疗效果优劣；探讨这三种微创治疗技术的作用机制及临床疗效；为临床上正确地选择这三种微创技术治疗肝癌提供理论依据和实验依据。方法取制作成功VX₂肝癌模型的新西兰大白兔（接种VX₂细胞株后3周）45只，采用螺旋CT增强扫描检查了解肿瘤大小及肝内转移、肺部转移、腹腔淋巴结转移和腹腔种植转移等情况后随机分为五组，每组9只，即氩氦刀冷冻组（A组）、射频消融组（B组）、微波凝固组（C组）、手术切除组（D组）和对照组（E组）。将实验兔用3％戊巴比妥钠（1ml／kg）全麻后固定于兔手术台上。在上腹部正中原伤口疤痕处作2—3cm长切口打开腹腔，暴露肝脏，用镊子轻夹肝左叶将其拉出体外，观察并估计肿瘤的大小。然后A、B、C组分别采用美国Endocare公司生产的氩氦刀冷冻系统、珠海市和佳医疗设备有限公司生产的HGCF—3000冷极射频肿瘤治疗机和南京庆海微波电子研究所生产的MTC—3C组织间肿瘤微波凝固治疗仪进行分组消融治疗，分别将氩氦刀针、射频电极针（单极冷循环）和微波电极针沿肿瘤长轴（纵径）直接穿刺肿块中心，针尖达肿瘤对侧边缘，然后进行消融；各组治疗时间均为该治疗设备用于人肝癌临床治疗时间的1／3（分别为5分钟、5分钟和3分钟）。D组行手术切除，切缘超过肿瘤边缘1.0cm。、E组开腹查看但不干预。五组处理完毕后，将兔肝左叶回纳入腹腔，关腹。在兔左后腿处肌注庆大霉素2万u，送回动物房饲养。观察各组生存期情况。各组兔死亡后均作尸检了解肿瘤残留情况及肿瘤转移情况，包括肝内转移、肺部转移、腹腔淋巴结转移和腹腔种植转移等。所有兔在穿刺接种前、治疗前及治疗后10天抽外周血，采用ELASA双抗夹心法检测可溶性白介素—2受体（sIL—2R），并检测丙氨酸转氨酶（ALT）。应用SPSS软件进行统计学分析。计量资料用均值±标准差。统计前经方差齐性检验；满足齐性，五组间比较用单向方差分析，两两比较用S-N-K法；若不满足齐性用Welch方法，两两比较用Games-Howell法。各组内治疗前后用配对t检验。P＜0.05有统计学意义。结果一、肿瘤残留及转移情况：1．肿瘤残留：A、B、C、D、E组肝内肿瘤残留分别有2、4、5、0、9只兔，残留率：E组＞C组＞B组＞A组＞D组，五组比较差异有显著性（x²=20.700，P=0.000）。2．肝内转移：A、B、C、D、E组肝内转移分别有1、3、4、6、9只兔，肝内转移率：E组＞D组＞C组＞B组＞A组，五组比较差异有显著性（x²=15.652，P=0.004）。3．肺部及腹腔淋巴结转移：各组全部兔都出现肺部及腹腔淋巴结转移。4．腹腔种植转移：A、B、C、D、E组腹腔种植转移分别有2、5、6、0、1只兔，腹腔种植转移率：C组＞B组＞A组＞E组＞D组，五组比较差异有显著性（x²=13.894，P=0.008）。B、C组腹腔种植转移率高可能与“煮沸效应”有关（即在行RFA和MCT治疗时可见煮沸的肿瘤组织液沿针道随蒸气流出进入腹腔，这些组织液可能含有活的肿瘤细胞）。上述结果提示：从肿瘤残留情况及转移情况上看，治疗兔VX₂肝癌，手术切除是最有效的方法，其次是氩氦刀冷冻，再其次是RFA和MCT。二、免疫功能变化：可溶性白介素—2受体（sIL—2R）可间接反映机体免疫力的变化，sIL—2R水平高低与机体免疫力强弱成反比。各组治疗前sIL—2R水平无显著性差异，治疗后氩氦刀冷冻组较治疗前有所下降，RFA和MCT组较治疗前无明显变化，手术切除组较治疗前有所上升，而对照组仍显著升高。说明上述四种治疗方法虽不能完全逆转荷瘤兔的抗肿瘤免疫抑制，但防止了如对照组兔的抗肿瘤免疫力的继续显著下降，使机体正常的免疫功能得到部分恢复。这种增强免疫力的作用以氩氦刀冷冻组最强，其次为RFA和MCT组，再其次为手术切除组。三、肝功能变化：各组间、组内比较，治疗前后ALT变化无显著性差异。说明氩氦刀冷冻、RFA、MCT和手术切除对实验兔肝功能影响不大。四、生存期情况：氩氦刀冷冻、RFA、MCT和手术切除组与对照组比较平均生存期有显著性差异（F=73.084，P=0.000）；氩氦刀冷冻、RFA、MCT和手术切除组平均生存期均显著高于对照组；氩氦刀冷冻和手术切除组显著高于RFA、MCT组；氩氦刀冷冻和手术切除组之间、RFA和MCT组之间平均生存期无显著性差异。这个结果提示我们：治疗兔VX₂肝癌，在延长兔生存期方面，氩氦刀冷冻治疗可取得与手术切除治疗相同的效果，显著高于RFA和MCT；四种治疗均显著高于对照组；而RFA和MCT比较无显著性差异。结论在使用氩氦刀冷冻、RFA、MCT治疗兔VX₂肝癌中：无论是在减少肿瘤残留和转移方面，还是在增强机体免疫力和延长实验兔生存期方面，氩氦刀冷冻治疗均优于RFA和MCT治疗，而RFA治疗和MCT治疗疗效相当；特别是在行RFA和MCT治疗时，由于存在“煮沸效应”（boiling effect），容易发生经针道的腹腔种植转移。上述结果值得引起临床关注。更多还原

【Abstract】 BackgroundLiver cancer is one of the most common malignant tumors and has rank in thirdin our country. Operation is the most effective treatment liver cancer in early stage.Due to its occult onset, the late visit to doctors combined with cirrhosis leading to badliver function, the rate of the surgical operation is only 20%-30%. For unsecetablepatients with medium and latter stage liver cancer, microinvasive treatment still canget the good curative effect. Therefore, microinvsive therapy has become anindispensable and important method in the treatments of the later period liver cancer.Frequently the microinvasive treatment to the liver cancer have two main type: one isangio-microinvasive treatment, including the transcatheter arterial infusion（TAI）,transcatheter artery embolization（TAE） and transcatheter arterialchemoembolization（TACE）;Another is none angio-microinvasive treatment,including percutaneous chemo-ablation and percutaneous physico-ablation, theformer includes percutaneous alcohol injection （PEI） and percutaneous acetic acidinjection （PAI）, and latter includes the cryocare^TM cryoablation, radiofrequencyablation （RFA）, microwave coagulation therapy （MCT）, laser mesenchymalthermotherapy and high intensity focused ultrasound. Among them, cryocare^TM cryoablation, radio frequency ablation and microwave thermotherapy are wiedlyapplied in the clinical. The three kinds of microinvasive therapy belong topercutenousphysico-ablation techniques, which can destroy or kill tumor cell by cold or hoteffect and attain" destruction in one time "therapeutic outcome." destruction in onetime " is defined that technique can destroy and kill tumor cell in the treatment scopein one time. Therefore, using these three kinds of microinvasive therapy in thetreatment of the liver cancer has strong comparability in its clinical curative effect.But for the three kinds of microinvasive therapy, which is the most excellent inclinical curative effect is not clear, and currently there is no the related experimentcontrast or the clinical contrast research in domestic and intemational. The model ofrabbit VX2 liver cancer which was established on the big animal is suitable for theexperimental study of liver cancer microinvasive treatment. VX2 tumor cell strain isoriginated from the squamous carcinoma that is derived from the rabbit’s papillomainduceing by Shope virus. Through seventy-two times transplantion and passage,VX2 tumor cells formally produce strain. At present, it is one of the best ideal livercancer experiment models. It is widespread applied to empirical study such as livercancer oncology, tumor therapeutics,radiobiology, pharmacokinetics of anticancerdrugs and tumor imageology. The purpose of this study is comparing respectivelygood and bad therapeutic efficacy by observing the size of tumor ablation target area,tumor cell necrosis and residue, tumor metastasis, and the change of immunologicalfunction as well as rabbit’s life span through putting the VX₂ liver cancer model ofrabbit into group with above-mentioned three kinds of microinvasive treatmenttechnique. To explore the mechanisms and clinical therapy of three micrioinvasivetechniques and provide theoretical and expermental bases for properly choosing themin liver cancer treatment in clinical.Chapter one The establishment of rabbitVN₂ liver cancer modelObjectivesIt has been proofed that microinvasive treatment is an important part in middle and advanced stage of the liver cancer, it is the first choice of nonoperative treatmentto the liver cancer. But now it still exits many problems, which include furtherimprovement of blood-supply theories, the prostecdtive efficacy and recurrence ofmicroinvasive treatment, the relationship between microinvasive treatment andapoptosis and drug resistant and so on. Moreover nonvascular treatment of eachsection remains to be studied such as the refrigeration, radio frequency, microwaveetc. A stable bigger animal model needs to be established. The ideal liver cancermodel should be the same or similar to liver’s biology behavior of human beings,which has the high achievement ratio of the model, lowmortality, good repeatability,convenient and animal size or figure suitable for the research of the etiopathogenisis,diagnosis and treatment. The method must be harmless or little harm to human bodyand little pollute the environment. The former tumor model establishes mostly in therat,due to it’s body is small and blood vessel is thin, the tumor is small, this methodis not suitable for using as the treatment research of.the interventional therapy of thetumor. The other animal is few used, such as pig and dog, because the lure livercancer of these two kinds of animals is different, the individual is difficult to compare;the achievement ratio of transplanting the liver cancer is low because of rejection andhard to live. But the figure of rabbit is bigger than rat, which is suitable for livercancer microinvasive treatment experimental research. The rabbit VX₂ liver cancermodel is suitable for the experimental study of liver cancer microinvasive treatment.Strain of VX₂ tumor cell is originated form the squamous carcinoma that is derivedfrom the rabbit’s papilloma inducing by Shope virus. Through seventy-two timestransplantion and passage, VX₂ tumor cells formally produce strain.The model ofrabbit transplantation VX₂ liver cancer has many advantages: （1）The model is thebiggest liver cancer model in china at present; （2）It has been demonstrated that tumorisrich in vascular, whose blood is provided by liver artery. The tumor is the similar to human primary hepatic carcinoma; （3） In pathology the tumor is similar to hugetype cancer of liver with huge lump solid tumor, infiltrate growth and plentiful bloodprovides; （4） Generally, the time needs three to four weeks for the rabbit VX₂ livercancer diameter grows to 3cm. So the rabbit VX₂ liver cancer model has been one ofthe most ideal liver cancer experimental models. It is widely applied to empiricalstudy such as liver cancer oncology, tumor therapeutics, radiobiology,pharmacokinetics of anticancer drugs and tumor imageology. The rabbit liver VX₂cancer model in this study was established via coeliotomy-inoculation method.MethodsLeft hepatic lobe is usually choosed for plantting cell line in rabbit VX₂ livercancer model.The methods include :（1） cut the belly open to embed; （2） cut the bellyopen to puncture; （3） direct puncture per cutem; （4） cell suspension innoculation. 80New Zealand big and white rabbits were established the model of liver tumors by cutthe belly open to puncture.This study ensured the inoculated location and the depthaccurate as far as possible, to increase the experiment comparability throughestablishing the rabbit liver VX₂ cancer model by the coeliotomy. This method wassimple, economic, high achievement ratio and accurate inoculation location, whichwas good for playing the microinvasive target ablation technique through planting thetumor to 1～3cm in 2～3 weeks.Results76 among 80 New Zealand rabbit models were built successfully and confirmedby CT and pathological examination .Inoculation rate was 95% in line with literaturereport.ConclusionsThe rabbit VX₂ liver cancer model is a ideal model for microinvasive treatmentstudy, whose inoculation method is simple, economic, high achievement and accurate inoculation location through planting the tumor to 1～3cm with 2～3 weeks.Chapter Two Comparison of ablation effect cryocare^TMcryoablation and radiofrequency ablation, microwavecoagulation therapy in rabbit with VX₂ liver cancerObjectivesThere are two kinds of microinvasive treatment in the clinical. One is an angio-microinvasive treatment, including the transcatheter arterial infusion chemotherapy（TAI）, transcatheter arterial embolzatization（TAE） and transcatheter hepatic arterialchemoembolization（TACE）. Another is non-angio-microinvasive treatment,including percutaneous chemo-ablation and percutaneous physico-ablation, theformer includes percutaneous alcohol injection （PEI） and percutaneous acetic acidinjection （PAl）, the latter includes the cryocare^TM cryoablation, radio frequencyablation （RFA）, the microwave coagulation therapy （MCT）,laser mesenchymalthermotherapy and high intensity focused ultrasound. Three kinds of the mostcommon microinvasive treatments in liver cancer include the cryocare^TMcryoablation、radio frequency ablation （RFA）, the microwave coagulation therapy（MCT）. They kill the tumor cell through cold or hot effect. The main biology curativeeffect of the cryocare^TM cryoablation treatment is the refrigeration that argongenerates. But the hot effective that argon created effects as well, because in a shorttime refrigeration and heat can create the ice crystal inside and outside the cell,damaging the tumor cell thoroughly. The mechanism by which cancer cell death thatdue to the super low temperature refrigeration is : 1. the cell dehydration andcrenulation; 2. the cell electrolyte toxicity condense and PH value change; 3.degeneration in the cell membrane lipoprotein composition; 4. mechanical damage of the formation and ice crystal in cells; 5. the stasis of blood stream andmicro-thrombosis; 6. the immunity response. Radio frequency ablation （RFA） andmicrowave coagulation therapy （MCT） belongs to scope of the hot ablation, whichkill the cancer cell using super heat, and the mechanisms of action are : 1.the tumororganizes congestion and hypoxia; 2. PH of the tumor reduces,the acidityincreases,lysosome increases,lysosome enzymelization; 3. the DNA and RNA proteinsynthesis of lump cell suffers from repression; 4. the organism immune reactionstrengthen .In the progress of using the three kinds of physico-ablation mircoinvasive,the size of the target area and the ability to kill the tumor cancer affect the therapeuticefficacy directly. The purpose of this experiment was to observe and compare thesize of ablation target area, cell death and residual situation via cryocare^TMcryoablation, radio frequency ablation and microwave coagulation therapy to meltthe rabbit VX₂ liver tumor.MethodsThe model of liver tumors were established in 27 New Zealand big and whiterabbits （inoculated VX₂ cell stub empresses successfully 3 weeks ago）.They wererandomizedly divided into 3 groups with 9 rabbits in each group, after using spiral CTto measure the perpendicular diameter and transverse diameter. Cryocare^TMcryoablation was A, the group of radio frequency ablation was B, microwavecoagulation was C. The rabbits were fixed on the operation table which wereanesthetized generally by 3% Pentobarbital （1mg/Kg）. The rabbits were cut the bellywith 2-3cm long cut through the abdomen median incision, then the tumor weremeasured through exposing the liver and extruding left hepatic lobe outside withtweezers. After that, the tumors were melted individually with the cryocare^TMcryoablation system of United States Endocare company, HGCF-3000 cool-tip radiofrequency ablation treatment machine of HeJia medical treatment equipments company in Zhuhai and MTC-3C microwave coagulation treatment appearance ofNanjing Qinghai mircowave electron institute. The each melt time is 1/3 the clinicaltreatment time patient with liver cancer. All the rabbits were killed after 3 days, thetumors were taken and fixed with Formaldehyde, then send for pathologicexaminination. All data was analyzed by SPSS software. Quantitative data wasexpressed by x±s, before statistics the test of homogeneity of variance were applied,ANOVA and S-N-Ktest or Welch and Games-Howell test in the comparison of threegroups were used. P＜0.05 were defined as statistical significance.Results1. The traverse diameter of ablation target area:that is 2.28+0.12cm,1.96±0.09cm and 1.93±0.10 cm in group A,BandCrespectively. There was significantdifference in the comparison of the three groups （F=31.138,P=0.000）and between Aand B,A and C（P=0.000）. There was no significant difference between B and C（P=0.653）.2. The average area in the ablation target area:that of A was 5.79±1.34 cm², Bwas 4.40±0.43 cm², C was 4.31±0.59 cm². The differences between Aand B,A and C（P=0.003,P=0.002 ）and differences among groups（F=8.047,P=0.002） are significant.No significant difference was found between B and C （P=0.836）.3. The rate of tumor complete ablation in Awas 88.9 %, 44.44 % in B, 33.44 %in C.Significant differences were seen in the comparisons of 3 groups（x²=6.300,P=0.043） and between A and B,A and C（P＜0.05）.There was no significantdifference between B and C （P＞0.05）.4. The tumor cell residual rate were 22.22 %, 77.78%,77.78% in A, B and Crespectively. There was significant difference in the comparison of the 3groups（x²=7.670,P=0.022）; There was significant difference in the comparison of thetumor cell residual rate between A and B, A and C. But there was no significant difference between B and C （P＞0.05）.5. The complete necrosis rate in A （66.67%） is higher than that of B and C（50.00%）.ConclusionsIn the three kinds of microinvasive treatment, cryocare^TM cryoablation wasbetter than RFA and MCT, no matter in the ablation target area and transversediameter, the rate of complete tumor ablation, or in the residual tumor rate. Howeverthe RFA equaled with the result of MCT. The study showed the effect of cryocare^TMcryoablation might be better than RFA and MCT in curing the rabbit VX₂ liver cancer.Chapter Three Comparison of therapeutic effect ofcryocare^TM cryoablation and radiorequency Ablation,microwave coagulation therapy in rabbits with VX₂ LivercancerObjectivesLiver cancer is one of the most common malignant tumors and has rank in thirdin our country. Operation is the most effective treatment liver cancer in early stage.Due to its occult onset, the late visit to doctors combined with cirrhosis leading to badliver function, the rate of the surgical operation is only 20%～30%. For unsecetablepatients with medium and latter stage liver cancer, microinvasive treatment still canget the good curative effect. Therefore, microinvsive therapy has become anindispensable and important method in the treatments of the later period liver cancer.Frequently the microinvasive treatment to the liver cancer have two main type: one isangio-microinvasive treatment, including the transcatheter arterial infusion（TAI）,transcatheter artery embolization（TAE） and transcatheter arterialchemoembolization（TACE）;Another is none angio-microinvasive treatment,including percutaneous chemo-ablation and percutaneous physico-ablation, the former includes percutaneous alcohol injection （PEI） and percutaneous acetic acidinjection （PAI）, and latter includes the cryocare^TM cryoablation, radiofrequencyablation （RFA）, microwave coagulation therapy （MCT）, laser mesenchymalthermotherapy and high intensity focused ultrasound. Among them, cryocareTMcryoablation,radio frequency ablation and microwave thermotherapy are wiedlyapplied in the clinical. The three kinds of microinvasive therapy belong topercutenousphysico-ablation techniques, which can destroy or kill tumor cell by cold or hoteffect and attain" destruction in one time "therapeutic outcome." destruction in onetime" is defined that technique can destroy and kill tumor cell in the treatment scopein one time. Therefore, using these three kinds of microinvasive therapy in thetreatment of the liver cancer has strong comparability in its clinicai curative effect.Three kinds of microinvasive therapy, which is the most excellent in clinicalcurative effect is not clear, and currently there is no the related experiment contrast orclinical contrast research in domestic and worldwide. In this experiment we createdindependently the rabbits VX₂ liver cancer with three kinds of microinvasive therapy,cryocare^TM cryoablation,radio frequency ablation,comparing with operational groupand the control group, then we told the result good from bad by the observationaltumor remained, tumor metabasis, the immunity function variety and the rabbitexisting period. We approached the function mechanism and clinical curative effectsof these three kinds of microinvasive therapies. In this way we can provide thetheories experiment accordance for the accuracy chance of the three kinds ofmicroinvasive therapies in the clinic treatment of the liver cancer.Methods45 New Zealand big and white rabbit（ inoculated VX₂ cell lines successfully 3weeks ago） were randomly divided into five groups （each group contains 9）, namelythe cryocare^TM cryoablation group（A group） radio frequency ablation group （B group）,microwave coagulation group （C group）, surgical operation group （D group） and thematched group （E group）,through using the spiral strengthened CT to scan check thetumor size and the transfusion in liver、lung and in the lymphoid node of the belly cavity. The rabbits were fixed on the operation table which were anesthetizedgenerally by 3% Pentobarbital （1mg/Kg）. The rabbits were resected from the bellywith 2-3cm long cut through the abdomen median incision, then the tumor weremeasured through exposing the liver and extruding left hepatic lobe outside withtweezers. After that, the tumors were melted respectively with the EndocareCryocare^TM Surgical System of United States Endocare company, HGCF-3000cool-tip radio frequency ablation treatment machine of HeJia medical treatmentequipments company in Zhuhai and MTC-3C microwave coagulation treatmentmachine of Nanjing Qinghai mircowave electron institute. The each melt time is 1/3the clinical treatment time in patient with liver cancer. Used the surgical operation tocuts off the tumor of the D group, and the incisal edge exceeded tumor edge 1.0 cm.Opened the stomach of the E group to look into it but did not interfere. Aftercompleting the five groups with different methods, retumed the left leaf liver into thebelly cavity, and sutured stomach. The rabbits were intramuscularly injected 20,000ugentamicin in left back legs, then send them back to the animal building for feeding.Observed them one. Made autopsy in each died rabbit to observed the remainedtumor and tumors transfusion, including the liver inside transfusion, the lungtransfusion, the implantation and the metastasis in lymphoid node in belly cavityand so on. All rabbits were taken the blood before inoculated, before treatment andtreated 10 days later, then used the ELASA bi-anti-sandwich method to examinesIL-2R and ALT. All the data was analyzed by SPSS software. Quantitative data wasexpressed by x±s, before statistics the test of homogeneity of variance were applied;ANOVA and S-N-K test in the comparison of three groups were applied. P＜0.05 weredefined as statistical significance.Results1. The tumor remained and metabasis: （1） The tumor remained inside the liver:2, 4, 5, 0 and 9 had tumor remainedinside the liver from the group A, B, C, D, E respectively. The residual rate was groupE＞group C＞group B＞group A＞group D.There was significant difference in thecomparison of the five groups （x²=20.700,P=0.000）.（2） The metabasis in liver: 1, 3, 4, 6 and 9 had metabasis in liver whose rate wasgroup E＞group D＞group C＞group B＞group A.There was significant difference in thecomparison of the five groups（x²=15.652,P=0.004）.（3） The metabasis in the lung and lymphonode of abdominal cavity:There were9 found metabasis in the lung and lymphonode of abdominal cavity in each group.（4） The carcinomatosis in abdominal cavity:The 2, 5, 6, 0 and 1 had thecarcinomatosis in abdominal cavity whose rate was group C＞group B＞groupA＞group E＞group D. There was significant difference in the comparison of the fivegroups （x²=13.894,P=0.008）. The high carcinomatosis rate of group B and C wererelated to the "boiling effect",that is during RFA and MCT treatment, boilinginterstitial fluid （and possibly viable tumor cells） emanated from the probe path intothe peritoneal cavity.The above results demonstrated: From the remained tumor and the metabasiswe concluded that the surgical operation is the most valid method, the next is thecryocare^TM cryoablation, the last are the RFA and MCT in the treatment of rabbit VX₂liver cancer.2. The immunity change: There was no significant difference in sIL-2R among thosegroups before treatment, it had a descent in the cryocare^TM cryoablation group, noobvious variety in the RFA and MCT group, a raise in the surgical operation group,and it showed significant advance in the matched control group, sIL-2R can reflectthe immunity change indirectly. Although above four kinds of treatment methodscouldn’t completely converse the anti- tumor immunity of the cancer rabbits, prevented the anti-tumor immunity from continuously descending, such as thematched control group and promoted the normal immunologic function partiallyrecovery. In the function of promoting immunity, the cryocare^TM cryoablation groupwas the best, the next was the RFA and MCT group, the surgical operation group wasthe third.3. The change of the live function:There was no significant difference in the ALT .Socryocare^TM cryoablation, RFA, MCT and surgical operation had no significanteffect on the live function of the rabbits.4. The mean survival time:That of the cryocare^TM cryoablation group, the RFA andMCT group and the surgical operation was notably higher that of matched controlgroup. There was significant difference in the comparison of the fivegroups（F=73.084,P=0.000）. The Cryocare^TM cryoablation group and the surgicaloperation group were higher than the RFA and MCT group; There was no significantdifference in the mean survival time between the cryocare^TM cryoablation and thesurgical operation group, this result can be seen on the RFA and MCT group.ConclusionsIn the treatment of the rabbit VX₂ liver cancer, the effect of the cryocare^TMcryoablation was superior than the RFA and MCT in the aspects of reducing tumorremains and metabasis, strengthening the immunity and prolonging the mean survivaltime. However the RFA equaled with the effect of MCT. During RFA and MCTtreatment, because occurrence of "boiling effect", the carcinomatosis in abdominalcavity is produceed easily. This result deserves to cause the clinical concerns.更多还原