【作者】 袁耒；

【导师】 徐志飞；

【作者基本信息】 第二军医大学 ， 外科学， 2008， 博士

【摘要】 第一部分改良型兔肺VX2肿瘤模型的建立目的:建立适合射频治疗的更理想的兔肺肿瘤模型。材料和方法:新西兰大白兔16只,CT引导下穿刺,将载有VX2肿瘤细胞悬液的P407凝胶接种于右肺内。结果:接种后10-14天,CT检查证实16只新西兰大白兔肺内有肿瘤生长,其中14只呈单发结节表现,直径0.8-1.0cm,兔肺VX2肿瘤模型成功率为100%;另有2只肺内呈多发结节生长,并出现胸腔积液;4只累及纵隔;2只出现胸壁种植转移。接种过程未出现气胸、出血等并发症。结论:本实验模型较以往模型操作简便,避免了悬液外溢导致的肿瘤弥散性生长,减少了并发症,可在肺内局部形成较大体积的实体瘤,能够满足射频治疗肺癌的研究需要,因此所建兔肺VX2肿瘤模型是成功的。第二部分活体兔肺的射频实验研究目的:探讨活体肺组织中在不同起始功率、不同电极直径条件下,射频消融效果的不同,选择适合消融动物模型的电极直径和功率时间组合。材料和方法:采用RF2000型射频治疗机,新型细径穿刺电极,打开最大直径2厘米,频率为465MHz,分别采用电极直径5mm,8mm,11mm,10w、20W、30W的起始功率,梯次增加功率,对正常兔肺射频消融,并用电极自带测温系统实时记录温度。结果:电极直径相同的情况下,不同起始功率,对应消融范围无明显差异,治疗最高温度无明显差异,但消融时间差异明显,起始功率越大时间越短,病理显示10 W起始功率时消融最彻底,30W起始功率时,虽然时间最短,但射频区凝固坏死间隔分布,消融效果不佳。结论:电极直径8 m可以满足兔肺肿瘤射频治疗需要,低起始功率较长的治疗时间射频消融效果最佳。第三部分高温与亚高温射频治疗兔肺内VX2肿瘤的对比研究目的:通过对普通高温射频消融方法的改进,寻找更能充分发挥抗肿瘤效应的射频治疗方法。材料和方法:50只新西兰白免体内建立肺癌模型,随即分为3组:对照组10只,高温射频组20只,亚高温射频组20只。观察动物生存期,射频治疗后肿瘤组织SDH的活性,HSP90的含量,以及静脉血T细胞亚群CD4、CD8、CD4/CD8的水平。结果:射频治疗后72小时两治疗组动物SDH水平均为阴性,亚高温射频组肿瘤组织HSP90明显高于高温射频组,亚高温射频组CD4、CD8、CD4/CD8水平的升高显著高于高温射频组,亚高温射频组动物生存时间也长于高温射频组。结论:亚高温射频治疗灭活肿瘤病灶可靠,同时可激发高水平的抗肿瘤免疫效应,治疗效果优于普通高温射频消融。第四部分树突状细胞对肿瘤射频灭活的辅助治疗作用目的:探讨射频治疗联合应用体外培养扩增的树突状细胞(DC)的抗肿瘤免疫增强作用。材料和方法:无菌状态下取兔骨髓造血干细胞,在重组兔粒细胞-巨噬细胞集落刺激因子、白细胞介素-4、肿瘤坏死因子α的刺激下,培养为成熟的树突状细胞。荷瘤兔随机分成3组:对照组10只,单纯射频组15只,射频+DC组15只。将成熟的树突状细胞注入亚高温射频治疗后的肺肿瘤病灶,观察各组动物病灶中树突状细胞的浸润情况,周围血IL-2、CD4、CD8水平CD4/CD8比值及动物的存活时间。结果:射频+DC组动物生存时间较单纯射频组延长,周围血IL-2、CD4水平CD4/CD8比值均显著升高。结论:射频治疗联合应用体外诱导培养DC细胞可进一步增强射频治疗所引起的抗肿瘤免疫反应,对兔肺VX2肿瘤具有一定的辅助治疗效果。更多还原

【Abstract】 PART ONE Establishment of Modified Rabbit Lung VX₂ TumorObjective:To establish more ideal rabbit lung tumor model suitable for radiofrequency.Materials and Methods:By CT guided,16 New Zealand white rabbits were implanted in right lung with P407 gels,which carried VX₂,tissue suspension.Results:Ten to fourteen days after implantation,tumors were found in 16 rabbits by CT scan.14 of them have single nodule in right lung,whose diameter was from 0.8-1.0 cm,The achievement ratio of model was 100%（16/16）.There were multi-nodule in 2 rabbits combined with pleural effusion.Mediastinum was involved in 4 rabbits.Implantation metastases in chest wall were found in 2 rabbits.Conclusion:The rabbit lung VX₂ tumor models are established successfully.The volume of solid tumor formed in lung is big enough for study of radiofrequency.The model is much easier to establish.It can avoid dispersing growth of tumor resulted by overflow of suspension,which reduces complication.PART TWO Experimental Study on Radiofrequency for Rabbit’s Lung in vivoObjective:To investigate radiofrequency ablation’s effect for rabbit’s lung in vivo with different electrode diameter at different original power output.Then select electrode diameter and power/time combination suitable for animal models.Materials and Methods:RF-2000 radiofrequency machine has a new thin-diameter puncturing electrode,which has a 465MHz frequency and a 2cm diameter when opened most.When ablating normal rabbit’s lung with electrode diameter of 5mm,8mm,11mm respectively and original power output of 10W,20W,30W additionally,temperature curves of selected spots were simultaneously recorded by electrode coupled temperature-measuring system.Results:With the same electrode diameter but different original power output,there’s no obvious difference in ablation area and highest temperature correspondingly.However, difference in ablation time was obvious,which appears the larger the power output was,the less time the ablation needed.As pathology shows,ablation was thoroughly when power output was 10W.When 30W,ablation effect was not quite well because of interval distribution of coagulation and necrosis in radiofrequency area,although ablation time is shortest. Conclusion:Electrode diameter of 8mm can meet the need of ablating lung cancer with radiofrequency.Best effect can be got with longer time at lower power output.PART THREE Compare Study on Thermal with Subthermal Radiofrequency Ablation for Rabbit Lung VX2 TumorObjective:To find out better radiofrequency method which is more effective to ablate tumors by improving common thermal radiofrequency treatment.Materials and Methods:50 New Zealand white rabbit lung VX2 tumor models were divided into 3 groups:thermal group（20）,subthermal group（20）,control group（10）. Observe their living time,activity of SDH in tumor tissue after radiofrequency ablation, content of HSP90 and count of CD4Tcells,CD8Tcells,CD4/CD8 ratio in venous blood.Results:SDH of two experimental groups showed negative 72hs after radiofrequency ablation.However,HSP90 content and CD4Tcells,CD8Tcells,CD4/CD8 ratio of subthermal group was markedly higher than that of thermal group.Meanwhile,living time of subthermal group was much longer.Conclusions:Subthermal radiofrequency ablation for tumor focus is safe and reliable,with a much better therapeutic effect than common thermal radiofrequency ablation.PART FOUR Accessory Effect of Dendritic Cells（DC） to Radiofrequency Ablation for TumorsObjective:To investigate the enhanced anti-tumor immunity of radiofrequency ablation combined with dendritic cells,which were cultivated in vitro.Materials and Methods:Activate the hemopoietic stem cells,which were got from normal rabbit’s bone marrow in aseptic environment,with GM-CSF,IL-4,TNFs from recombinant rabbit.Then cultivate the stem cells to mature dendritic cells.Divide 40 tumor rabbits into 3 groups:simple radiofrequency group（15）,radiofrequency and DCs combination group（15） and control group（10）.Inject equal quantity of saline fluids,mature DCs fluids, mature DCs fluids into the 3 groups respectively into lung tumor focus treated by subthermal radiofrequency ablation.Observe infiltration of DCs in tumor focus,content of IL-2,CD4Tcells,CD8Tcells,CD4/CD8 ratio in peripheral blood and living time of the animals.Result:Living time of radiofrequency and DCs combination group was longer than that of simple radiofrequency group.Content of IL-2,CD4Tcells,CD8Tcells,CD4/CD8 ratio in peripheral blood of radiofrequency and DCs combination were markedly higher.Conclusion:Radiofrequency combined with DCs induced in vitro can enhance anti-tumor immunity of radiofrequency therapy.That means DCs does have accessory effect for rabbit lung VX2 tumor.更多还原