【作者】 刘建刚；

【导师】 张积仁；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2010， 博士

【摘要】 肿瘤是目前世界上死亡率最高的疾病之一,严重威胁着人类的生命。然而,70%-80%的患者确诊时已属晚期,失去了手术治疗机会；而手术治疗,术后复发和转移率仍高达50%-70%,总的治愈率不足10%。因而,以灭活肿瘤细胞、减轻肿瘤负荷为目的的微创局部消融治疗技术近年来引起了广泛的关注,局部消融治疗可以为失去常规手术根治性治疗机会的实体肿瘤患者提供新的治疗选择。1998年10月美国Endocare公司研制成功一种新型超低温介入冷热消融氩氦靶向肿瘤治疗设备：Endocare CryocareTM Surgical System(简称美国氩氦刀),它的发明是冷冻热治疗技术发展的最新成就,不但继承发展了超低温治疗学的基础和临床研究成果,而且推出了肿瘤微创治疗的新概念—氩氦靶向肿瘤治疗技术(Cryocare Targeted Cryoablation Therapy)。广州南方医科大学珠江医院1999年在亚洲率先引进这项技术,为中晚期肿瘤患者治疗带来了福音。美国氩氦刀是目前唯一可进行经皮冷热消融治疗的先进技术。对于不能常规手术根治性切除肿瘤病人的治疗,应该首先选择微创消融治疗,微创消融快速减轻肿瘤负荷,对于减轻病人痛苦,提高生存质量,保证综合治疗疗效具有放化疗所不能代替的地位。在微创消融治疗技术当中,氩氦刀冷冻治疗创伤小,疗效好,有效率可以达到90%以上,已成为许多临床医生术中治疗不可缺少的手段之一。由于氩氦刀靶向消融治疗不需要剥除冷冻后的肿瘤组织,手术操作简便,患者术后并发症少,多数不影响其它治疗措施的选择。氩氦刀联合放疗、光动力治疗、中药治疗均可提高治疗效果。同时,实验和临床研究证实,冷冻治疗肿瘤可增强机体的免疫水平,有助于降低肿瘤的转移率和复发率。氩氦刀治疗有抗肿瘤免疫作用：主要是通过肿瘤细胞坏死,使肿瘤细胞分泌的免疫抑制分子减少,冻融坏死的原位肿瘤细胞可成为肿瘤抗原,而达到增强免疫的效果。1967年Yantorno等和1968年Shulman等首先证实冷冻免疫反应的存在,为冷冻免疫学奠定了基础。他们推测在冷冻治疗后,由于机体清除被摧毁的肿瘤组织,吞噬细胞递呈抗原产生针对残余肿瘤的抗体,包括产生针对肿瘤特异性膜表面蛋白的抗体,这些抗体结合到其他活的肿瘤细胞的膜表面蛋白上,引发补体固定和巨噬细胞及中性粒细胞的趋化作用,导致宿主的免疫系统被激活,使得剩余的肿瘤组织及远处转移瘤变小,这种反应被称之为冷冻免疫学效应。国内张国强等分别检测10例肺癌患者术前、后不同时期的外周血T淋巴细胞亚群及血清免疫球蛋白的改变,结果表明：冷冻术后CD3+T、CD4+T、CD4+T/CD8+T比值较术前显著增加,而CD8+T则显著减少,免疫球蛋白也显著增加。彭秋平等于肝癌患者中也观察到了CD3+T、CD4+T、CD4+T/CD8+T类似变化,但CD8+T细胞较术前则无明显变化。段蕴铀等则发现：氩氦刀冷冻处理的肺癌细胞可以致敏树突状细胞,当联合IL-2时,可提高NK细胞及T淋巴细胞的抗肿瘤活性。目前关于氩氦冷冻对免疫功能的影响缺乏基础实验的研究,要想阐明冷冻免疫的机制,也需要对更多的肿瘤细胞系进一步实验。临床上对于氩氦冷冻治疗肿瘤的免疫学效应认识不清,对氩氦冷冻治疗后是否需要协同免疫治疗亦未有明确认识。因此,有必要对氩氦冷冻的免疫学效应从基础实验到临床应用方面做进一步研究。本研究采用SD大鼠皮下移植瘤模型进行氩氦冷冻实验,探讨氩氦冷冻后大鼠局部和机体免疫功能的变化。观察氩氦冷冻后局部组织冷冻消融规律并对冷冻周围免疫杀伤区的存在进行了证实,通过检测氩氦冷冻后大鼠外周血T细胞亚群、sIL-2R含量及单个核细胞的杀伤能力,对氩氦冷冻对大鼠免疫功能的影响进行了深入的探讨。同时,通过检测76例病人氩氦冷冻前后外周血T细胞和NK细胞水平高低,探讨了氩氦冷冻联合中药治疗肿瘤的临床疗效及其临床意义。本研究共分为3部分：第一章SD大鼠皮下移植瘤模型的建立目的建立SD大鼠W256细胞皮下移植瘤模型,为氩氦冷冻治疗肿瘤提供一个理想的动物实验模型。方法w256细胞冻融后传代,调整细胞浓度后接种于大鼠腹腔,建立大鼠的腹水模型。以浓缩的腹水接种于大鼠右后腿皮下,建立大鼠的皮下移植瘤模型。定期测量肿瘤大小。于接种两周后切取长势良好肿瘤2个,并于试验结束后切取所有肿瘤,行HE染色,光学显微镜观察。结果所有接种SD大鼠全部接种成功,生长良好,肿瘤周围无感染病灶。肿瘤接种第4天起可扣及皮下小结节,到接种第3周左右肿瘤平均体积已经达到了3.5cm3,最大肿瘤长径达到5.5cm。2周后切取的肿瘤结节大体观察和HE染色,病理观察肿瘤为多个灰白色结节,质软,光学显微镜下癌细胞排列密集,细胞间无间质,瘤细胞呈团状或片状排列,核大,核仁明显,核分裂相多见,肿瘤内可见丰富的小血管。实验结束时肿瘤内均见不同程度片状坏死。结论采用浓缩腹水注射法制作SD大鼠w256皮下移植瘤模型,该方法简单、经济,直视下接种部位准确,成瘤率高,种植2-3周,肿瘤体积可至3.5cm3,利于微创靶向消融技术的实施,可为后期采用氩氦冷冻治疗肿瘤的实验提供理想的动物模型。第二部分SD大鼠皮下移植瘤氩氦冷冻治疗前后免疫功能的变化目的观察氩氦刀冷冻消融治疗对SD大鼠皮下移植瘤组织坏死和细胞凋亡的影响以及冷冻前后其免疫功能的变化情况,以进一步探讨氩氦刀冷冻消融治疗肿瘤的免疫学效应,为临床应用提供新的理论依据。方法1.将B组40只实验大鼠随机分成4组,分别为：荷瘤对照组,冷冻治疗组,手术治疗组,空白对照组(n=10),麻醉后分别处理。冷冻治疗组：氩氦冷冻皮下移植瘤,时间3-5min,复温时间30s。手术治疗组：将肿瘤尽量全部剥离后,创口清洁缝合。荷瘤对照组：将氩氦刀置入肿瘤3分钟,而不予冷冻治疗。正常对照组：只切开皮肤,然后清洁缝合。各组SD大鼠均在治疗前1天、治疗后第1、3、5周眼眶采血1.5ml,流式细胞术检测行T细胞亚群(CD3+T、CD4+T、CD8+T)、双抗体夹心法测血清sIL-2R的含量。2.将A组36只大鼠随机分为两组,即氩氦冷冻组和荷瘤对照组(n=18)。分别于冷冻后3,12,24 h和3d,,7d时处死动物,取材。每组各时间点各处死小鼠3只,尽量完整剥离肿瘤组织。剥离的肿瘤组织贮存于10%福尔马林溶液中,用于组织病理学学检查和免疫组织化学检测相关指标。结果1.氩氦刀冷冻消融肿瘤坏死情况氩氦刀冷冻消融治疗SD大鼠皮下移植瘤,冷冻后12h观察冷冻靶区可见三个比较明显的分区：冷冻区中心(围绕氩氦刀探头插入处,冰球中心区域,一般低于-38℃)细胞完全坏死崩解,核碎裂,呈凝固性坏死。坏死区域周边(冰球边缘)出现明显的损伤区带,其中仍可见部分癌细胞,但细胞皱缩,核浓集,染色质固缩,呈典型的凋亡细胞特征；局部血管充血、栓塞、渗出；外围则是未受损的肿瘤细胞,其间有大量的炎细胞浸润。2.TUNEL末端标记法原位检测细胞凋亡对SD大鼠皮下移植瘤实施局部冷冻后,TUNEL染色结果显示,凋亡细胞主要集中分布在冰球周围损伤区带,细胞出现了凋亡特有的形态学变化：表现为细胞核着棕黄色,核固缩,染色质浓缩,部分细胞已经破碎溶解,对照组肿瘤组织中少量凋亡细胞散在分布,与荷瘤对照组相比,冷冻组各时间点凋亡细胞阳性率均显著高于荷瘤对照组(P=0.000)。冷冻组凋亡细胞于术后3h即开始增多,术后12h凋亡达高峰(67.25±5.51)%。3.冷冻术后单个核细胞的杀伤能力冷冻后氩氦冷冻组其外周血单个核细胞的杀伤能力在各时间点明显增强,显著高于其它各组(P=0.000),正常对照组虽较荷瘤对照组和手术治疗组杀伤能力高,但统计学并无差异。4.T细胞分类检验及血清sIL-2R含量的测定结果①荷瘤组大鼠的CD3+T、CD4+T细胞的百分率以及CD4+T/CD8+T比值与正常对照组相比较有显著差异(P=0.000),且随时间延长而逐步呈降低趋势。手术组CD3+T、CD4+T细胞的百分率以及CD4+T/CD8+T比值随时间延长而呈上升趋势,与正常对照组相比较有显著差异(P=0.000),且术后与荷瘤组比较差异性显著(P=0.000)。冷冻组CD3+T、CD4+T细胞的百分率以及CD4+T/CD8+T比值同样随时间延长而呈上升趋势,且比手术组上升更明显(P=0.000)。②荷瘤组大鼠的CD8+T细胞的百分率在各个时间点与正常对照组相比较均有显著差异(P=0.000),且其值随时间延长而逐步呈增加趋势。手术组和冷冻组CD8+T细胞的百分率随时间延长而呈降低趋势,术后第3、5周与荷瘤组比较差异性显著(P=0.000)。③荷瘤组大鼠的外周血液中sIL-2R含量在各个时间点与正常对照组相比较有显著差异(P=0.000),且随时间延长而逐步呈增加趋势。手术组外周血液中sIL-2R含量随时间延长而呈降低趋势,术后第3、5周与荷瘤组比较差异性显著(P=0.000),且在各个时间点与正常组比较差异性显著(P=0.000)。冷冻组外周血液中sIL-2R含量同样随时间延长而呈下降趋势,且比手术组下降更明显,术后第3、5周与手术组比较差异性显著(P=0.000)。结论1、氩氦刀冷冻治疗不但有冷冻消融区还存在周围的免疫杀伤区,氩氦刀可通过坏死和凋亡两种途径达到有效的消融。氩氦冷冻治疗肿瘤要注意尽量把肿瘤包裹于冰球内,部分不规则的肿瘤可能因为冷冻消融的不完全而复发,因此冷冻治疗有必要与放化疗、免疫治疗以及其它的肿瘤治疗手段相结合。2、氩氦冷冻可有效的启动机体的免疫效应。冷冻后科刺激肿瘤相关抗原的持续释放,具有对肿瘤相关抗原特异性细胞毒性T细胞的激活、增殖作用,可有效诱发、增强机体的抗肿瘤免疫；并且可增强外周血单个核细胞的杀伤能力。3、氩氦冷冻对机体抗肿瘤免疫增强作用可能是有一定限度的,如何促进和维持氩氦冷冻的免疫调节作用将是一个值得进一步探讨的问题。第三部分氩氦冷冻联合中药治疗肿瘤对机体免疫功能的影响及其临床意义的研究目的采用氩氦刀联合中药治疗76例中晚期肿瘤的患者,以进一步研究氩氦冷冻治疗肿瘤对机体免疫功能的影响,并观察氩氦冷冻联合中药治疗的临床疗效,探讨氩氦冷冻联合中药治疗肿瘤机体免疫功能变化的临床意义。方法1.76例肿瘤患者均经病理诊断为Ⅲ期或Ⅳ期的晚期恶性肿瘤,随机分为2组,即氩氦冷冻治疗组和氩氦冷冻联合中药治疗组,每组38例。2.各组患者分别于治疗前及治疗后2周、4周空腹抽取肘静脉血经流式细胞仪测定T淋巴细胞亚群(CD3+T、CD4+T、CD8+T)分布和CD4+T/CD8+T比值,并检测NK细胞水平。结果76例患者氩氦冷冻治疗后CD3+T、CD4+T、CD4+T/CD8+T、NK细胞水平明显高于治疗前,其差异有显著意义(P=0.000)。氩氦冷冻联合中药治疗组在治疗2周后CD3+T、CD4+T、CD4+T/CD8+T、NK细胞水平略高于单纯氩氦冷冻组,但其差异无统计学意义(P>0.05)；在治疗4周后CD3+T、CD4+T、CD4+T/CD8+T、NK细胞水平明显高于单纯氩氦冷冻组(P=0.000),其差异有统计学意义。结论氩氦冷冻治疗可刺激机体免疫能力的提高,有利于解除晚期肿瘤患者的免疫抑制,提高患者的免疫水平。冷冻后联合应用驱邪扶正中药能够更好的提高机体的免疫力,提示氩氦冷冻联合中药等免疫调节剂将会在中晚期肿瘤的临床治疗方面开辟新的局面,对缓解患者痛苦,提高生活质量,减少肿瘤复发有重要意义。更多还原

【Abstract】 Cancer, a disease which has the highest mortality in the world, threatens human’s life.70-80% patients are already in the advanced stage when diagnosed, losing the chance of surgery. However, the total curative ratio of surgery is less than 10% with a postoperative recurrence and metastasis rate of 50-70%. Cryotherapeutic ablation therapy, targeting killing cancer cells to eliminate tumor load, provides a new choice for patients with solid tumor who have lose the chance of radical surgery.Endocare CryocareTM Surgical System, invented by Endocare corporation in October 1980 based on clinical and basic research in ultralow temperature therapy, is a new therapeutic equipment for ultralow temperature intervened Ar-He targeted thermal ablation, which introduced a new concept in minimal surgery in curing cancer- Cryocare Targeted Cryoablation Therapy. It is brought in by Zhu-Jiang Hospital of southern medical university in 1999. Endocare Cryocare Surgical System is an advanced technique which is the only one that can undergo percutaneous thermal cryoablation therapy. Minimal invasive ablation, which can quickly relieve tumor load to elevate quality of life and guarantee the effect of comprehensive therapy, is the first choice for patients losing the chance of radical surgery and can’t be taken placed by chemo and radioactive therapy. Endocare Cryocare Surgical System, which brings little wounds and better curative effect, is an important technique with an effective rate higher than 90%.Frozen tumor tissues are not removed in cryocare targeted cryoablation therapy so the operation is simple which brings less postoperative complications and has no effect on choosing other therapies. The curative effect of cryocare targeted cryoablation therapy can be evaluated when combined with radiotherapy, photodynamic therapy and traditional Chinese drug. It is also demonstrated by research that immune function can be enhanced to decrease recurrence and metastasis rate using cryotherapy. Cryocare targeted cryoablation therapy can enhance anti tumor immune function by inducing necrocytosis of cancer tissue to decrease immunosuppressive molecules secretion while necrotic in stiu cancer cells become tumor antigen.Immune response to cryotherapy is demonstrated by Yantorno in 1967 and Shulman in 1968 who settle the foundation of cryoimmunology. They inferred that antibodies targeting remaining tumor, including antibodies targeting tumor specificity membrane protein are produced after phagocyte present antigen when necrotic tumor tissues are cleared. These antibodies later bind to other membrane protein of alive tumor cells to induce complement fixation and chemotaxis of macrophagus and neutrophil to activate immune response and eliminate remaining tumor tissue and metastatic tumor. This procedure is called cryoimmnue response. Zhang Guo-Qiang et al detect the change of T-lymphocyte subpopulation in peripheral-blood and the serum levels of immunoglobulins in 10 patients before and after surgery, finding CD3+, CD4+, CD4+/CD8+ and immunoglobulins are significantly increased with CDT8+ decreasing significantly after cryosurgery. Peng Qiu-Ping et al found similar change of CD3+T, CD4+T and CD4+T/CD8+T in patients with hepatic cancer but no significant change in CD8+T cells level. Duan Yun-You et al reported that lung cancer cells treated with cryocare targeted cryoablation therapy could sensitize dendritic cells and anti tumor activity of NK cells and T lymphocytes when combined with IL-2. Researches of basic experiments on effect of cryotherapy on immune function are lacked so the mechanism of cryoimmnuology remains unclear needing more tumor cell lines experimented to be clarified. Anti tumor immune function of cryocare targeted cryoablation therapy and whether immunotherapy should be combined remain unclear in clinical practice. And thus, a summary of immune function of cryocare targeted cryoablation therapy from basic experiment to clinical practice is necessary.Subcutaneous xenotransplanted tumor model in SD rats were used in this study to undergo cryotherapy experiment to explore the change of local and overall immune function of SD rats. Principle of the cryoablation of local tissues is discussed and the presence of immune cytotoxicity in tissues around the cryotherapy ones is demonstrated. The effect of argon-helium cryoablation on immune function of SD rats is thoroughly explored through detecting the level of T-lymphocyte subpopulation and sIL-2R in peripheral-blood and cytotoxicity of single nucleus cells. The level of T-lymphocytes and NK cells in peripheral-blood of 76 patients before and after cryotherapy was detected to discover the clinical significance of argon-helium cryoablation combined with traditional Chinese drugs.This study includes three parts.Part 1:Establishment of subcutaneous xenotransplanted tumor model in SD ratsObjectiveTo establish subcutaneous xenotransplanted tumor model of W256 cell in SD rats to offer a animal model for argon-helium cryoablation.MethodsW256 cells were inoculated to enterocoelia of SD rats with proper concentration after being unfrozen to passage culture to establish mouse model of ascites. Concentrated ascites was inoculated in right rear of rats subcutaneously to establish subcutaneous xenotransplanted tumor model. Two tumors were removed two weeks after inoculation and all tumors removed at the end of experiments to undergo HE staining and be observed under light microscope.ResultsInoculation in 66 SD rats was successful with all rats growing well and no infected focus around tumors. Subcutaneous nodules could be percussed 4 days after inoculation. The average size of tumor was 3.5cm3 about 3 weeks after inoculation with the longest major axis of 5.5cm. Tumor nodules were removed 2 weeks after inoculation to undergo gross inspection and HE staining. Tumors were made up of multiple soft grey white nodules. Tumor cells, whose nuclei were large with clear nucleolus and multiple mitotic figures under light microscope, were tightly packed in a mass or compact sheet with no intercellular matrix between and abundant small vessels. Patchy necrosis were observed in tumors at the end of experiment.ConclusionEstablishment of subcutaneous xenotransplanted tumor model of W256 cell in SD rats using the method of injecting concentrated ascites is simple and economical with precise inoculation and high tumor proliferating rate. It’s easier to undergo minimal invasive targeting ablation with the size of tumor can be 3.5 cm3 2-3 weeks after inoculation. In a word, this animal model is perfect for experiments using argon-helium cryoablation to treat cancer. Part 2 Change of immune function in subcutaneous xenotransplanted tumors of SD rats after Argon-Helium CryoablationObjectiveTo observe the effect of argon-helium cryoablation on the tissue necrosis and cell apoptosis as well as the immune function in subcutaneous xenotransplanted tumor of SD rats to further discuss the immune function effect of argon-helium cryoablation in treating cancer and supply new theory for clinical practice.Methods1, Rats were divided randomly into 4 groups:cancer control group(A), cryoablation group(B), surgery group(C), normal control group(D) (n=10). They were treated after anesthesia as follow. Cryoablation group:subcutaneous xenotransplanted tumors underwent argon-helium cryoablation for 3-5 minutes and then rewarmed for 30 seconds. Surgery group:tumors were removed with wounds cleared and seamed. Cancer control group:Cryocare surgical system was put into tumor for 3 minutes with no cryotherapy. Normal control group:dermotomy were operated with wounds cleared and seamed.2, Rats were killed with tumor tissues removed 3,12,24 hours and 3,7,14 days after cryoablation.3 rats from each group were killed with tumor tissue removed thoroughly at each time point. Tumor tissues were stored in 10% formaldehyde solution for histopathology observation and detection of immunohistochemistry indexes.1.5 ml blood of each SD rats from each group were collected from venous sinus of eye-orbit 1 day before treatment and 1,3,5 weeks after treatment to detect T-lymphocyte subgroup (CD3+T、CD4+T、CD8+T) via flow cytometry and level of serum sIL-2R by double antibody sandwich method. Result1,Necrosis of tumors with argon-helium cryoablation Three obvious zones of focus could be seen after subcutaneous xenotransplanted tumors underwent argon-helium cryoablation. It could be seen after cryotherapy at the center of focus (area around the place where probe were inserted) that cells were completely destructed with broken nucleic and coagulative necrosis. Obvious damaging area around necrosis could be seen with some tumor cells which had typical characteristics of apoptosis cells with cell shrinkage and condensation and margination of nuclear chromatin. Vessels at the area were congested, embolized and effusing. Tumor cells at the peripheral area were not damaged and had normal morphology.2, Detection of Cell Apoptosis by TUNEL Subcutaneous xenotransplanted tumors tissue was observed with TUNEL staining after argon-helium cryoablation. Apoptosis cells with typical apoptosis characteristic, whose nucleic were brown and pyknotic with concentrated chromatin and some broken cells mainly gathered in damaging areas around ice balls. There were few apoptosis cells in control group. Compared to cancer control group, positive rate of apoptosis cells in cryoablation group at each time poin was significantly higher(P=0.000). Apoptosis cells increased 3 hours after cryoablation, reaching the peak at 12 hours after(67.25±5.51)%.3, Cytotoxicity of Mononuclear cell after cryoablation Cytotoxicity of mononuclear cell was significantly enhanced after cryoablation, which is significantly higher in cryoablation group(P=0.05). Cytotoxicity of cells in normal control group was higher than that in tumor control group and surgery group but had no statistically difference.4, Detection of T-lymphocyte Subgroup and Serum sIL-2R levelCompared to normal control group, there is significant difference in percentage of CD3+T, CD4+T cells and CD4+T/CD8+T ratio which eventually decreased over time in cancer control group(P=0.000). Percentage of CD3+T, CD4+T cells and CD4+T/CD8+T ratio in surgery group which eventually increased over time was significantly different compared to normal control group(P=0.000). Percentage of CD3+T, CD4+T cells and CD4+T/CD8+T ratio in cryoablation group which also increased over time had significantly increased compared to surgery group(P=0.000).②Percentage of CD8+T cells in cancer control group which increased over time was significantly different compared to normal control group at each time point. Percentage of CD8+T cells in surgery group and cryoablation group which decreased over time was significantly different compared to cancer control group 3 and 5 weeks after treatment.③sIL-2R level of peripheral blood of rats in cancer control group which increased over time was significantly different at each time point compared to normal control group(P=0.000). sIL-2R level of peripheral blood of rats in surgery group which decreased over time was significantly different 3 and 5 weeks after treatment compared to cancer control group(P=0.000). sIL-2R level of peripheral blood of rats in cryoablation group which decreased more rapidly than that in surgery group over time was significantly different 3 and 5 weeks after treatment compared to surgery group(P=0.000).Conclusion1, That cryoablation area as well as peripheral immune cytotoxicity together indicated that argon-helium cryoablation took effect through necrosis and apoptosis. Tumor tissue should be completely packed in the ice ball when using argon-helium cryoablation. Some tumors with irregular shape may reoccur with incomplete cryoablation and thus argon-helium cryoablation should be combined with chemotherapy, radiotherapy, immunotherapy or other anti tumor methods. 2, Argon-helium cryoablation could effectively activate immune response. Secretion of tumor associated antigen was released continuously after argon-helium cryoablation, leading to the activation and proliferation of T-lymphocyte with tumor associated antigen specificity cytotoxicity which activated and enhanced anti tumor immune fuction. Cytotoxicity of mononuclear cells in peripheral blood was also increased, which might be higher when combined with cytokine.3, There is limitation in the enhancement of anti-tumor immune fuction by argon-helium cryoablation. So further exploration should be made in how to promote and maintain immunoregulation of argon-helium cryoablation.Part 3 Effect of Ar-He Targeted Cryoablation Combined with Traditional Chinese Drugs on Immune function and its clinical significanceObjectiveTo observe the effect of Ar-He targeted cryoablation combined with traditional Chinese Drugs on immune function and clinical outcome and its clinical significance.Methods1,76 patients pathologically diagnosed to have III or IV stage malignant advanced cancer were divided into two groups:argon-helium cryoablation group and argon-helium cryoablation combined with traditional Chinese drugs group, each 38 patients.2, Fasting venous blood from the patients’elbow were drawn 2 and 4 weeks before or after treatment to detect the subgroup of T-lymphocytes (CD3+T、CD4+T、CD8+T), CD4+T/CD8+T ratio and NK cell level via flow cytometry.ResultsLevel of CD3+T、CD4+T、CD4+T/CD8+T、NK cells of 76 patients was significantly higher after argon-helium cryoablation (P=0.000). Level of CD3+T、CD4+T、CD4+T/CD8+T、NK cells of patients in argon-helium cryoablation combined with traditional Chinese drugs group was slightly higher 2 week after therapy than that in argon-helium cryoablation group but had no statistics significance but 4 weeks after was significantly higher with statistics significance(P=0.000).ConclusionArgon-helium cryoablation can release immnosuppression in patients with advanced cancer while enhancing immune function. But there is limitation in the effect of argon-helium cryoablation on immune function and thus traditional Chinese medicine should be combined. Combination of these two therapy will decrease the recurrence of tumor, relieve patients’suffering and improve quality of life更多还原