【作者】 全毅；

【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】 研究背景原发性肝癌(HCC)的发生发展是一个多因素、多阶段、多基因变异的复杂过程,与机体的免疫状况密切相关,尤其是与T细胞的细胞免疫有关,但受多种因素的调节,肿瘤患者的机体免疫功能不同程度地发生紊乱。多数学者认为在机体的抗肿瘤免疫中细胞免疫占主导地位,研究表明,T淋巴细胞和NK细胞的比例及功能变化与肿瘤的发展、扩散有明显的相关性。有研究表明HCC患者外周血中CD3,CD4降低,CD8增高,免疫功能低下,恶变细胞所产生的免疫抑制是肿瘤患者免疫功能低下的主要原因。肿瘤免疫主要与T淋巴细胞的细胞免疫有关,T淋巴细胞亚群是反映机体细胞免疫功能的一个指标。T淋巴细胞是由不同亚群组成的,外周血T细胞亚群在数量上的协调比例,特别是CD4/CD8的比值是反映细胞免疫平衡与否的敏感指标,有助于反映机体免疫反应的调节能力。其中CD4+T淋巴细胞一般认为处于细胞免疫的核心,以其主要分泌细胞因子调节肿瘤免疫被称之为辅助性T淋巴细胞(Th),Th是人类主要组织相溶性抗原(MHC)Ⅱ类分子限制性T细胞,由于大多数肿瘤细胞不表达MHCⅡ类分子,故此类分子不能直接识别肿瘤细胞,而是依赖抗原提呈细胞如B细胞、巨噬细胞提呈相关抗原,对之进行特异性激发后,才分泌淋巴因子(如IL-2、r-干扰素等),激活B细胞、巨噬细胞、NK细胞,并通过其活化的细胞发挥抗肿瘤作用。Ts细胞(suppressor T cell)即抑制性T细胞在宿主免疫机制中起着复杂的抑制调控作用。当发生肿瘤后,Ts在功能和数量上都显著增强,抑制机体对肿瘤的免疫效应,从而使肿瘤逃逸机体的免疫监视,促进了肿瘤的生长。大量体外实验证实,Th和Ts细胞在正常情况下保持着恒定的比值,某一亚群变化可直接影响到群体功能,两者相互制约构成机体重要的免疫调节机制。肝癌的发生发展也同时与细胞凋亡存在着密切的关系,体外培养的肝癌细胞和体内发生的肝癌细胞均存在着细胞凋亡现象。未经治疗的肝癌会发生自发性癌细胞凋亡,恶性程度越高凋亡指数越大。肝癌时肿瘤细胞的增殖指数与凋亡指数呈正相关,从正常肝脏、癌前病变到肝癌,凋亡率随增殖率增高而增高,但其增高的幅度明显小于细胞增殖增高的幅度。肝癌组织中肿瘤细胞凋亡明显高于正常肝组织及癌周肝组织。细胞凋亡在肿瘤的发生发展中可能具有双面作用：细胞凋亡可能抑制癌细胞生长；也可能通过凋亡筛除那些生命力低下或表型类似正常细胞的肿瘤细胞,而选择侵袭力强增殖旺盛的细胞克隆,促进肿瘤发生发展。所以研究细胞凋亡的机制及凋亡在疾病过程中的作用,不但可以了解肿瘤发生发展的机理,更重要的是从一个全新的角度认识肿瘤细胞的生物学特性,为肿瘤治疗开辟新的途径提供了重要的理论依据。选择经肝动脉化疗栓塞术(transcatheter arterial chemoembolizaton, TACE)是不能切除肝癌公认的首选治疗方法,经肝动脉化疗栓塞治疗肝癌后,肿瘤细胞被杀死,肿瘤组织出现凝固性坏死,肿瘤细胞负荷减少,使肿瘤释放的TNF减少,免疫抑制因子的产生亦大大减少,减弱了对机体的免疫功能的影响,机体免疫细胞指标有所改善。经手术和栓塞治疗后肝癌患者的免疫功能受抑减轻。但肝动脉化疗栓塞治疗常包括栓塞和化疗两部分,化疗则抑制机体的免疫功能,所以对机体免疫功能的影响颇为复杂。另一方面,一般认为TACE作用机制是局部化疗和血管栓塞引起了肿瘤组织细胞坏死,但近年来发现栓塞引起的缺血缺氧是肿瘤细胞凋亡强有力的基因诱导动因,缺血数分钟就有即刻早期基因表达,缺氧也可以诱导凋亡基因表达增高,缺血至少可诱导80种以上的基因表达,包括促进细胞凋亡基因表达增高,抑制凋亡基因表达下降,使未凝固坏死的肝癌肿瘤细胞发生凋亡。因此,凋亡在TACE治疗肝癌所引起的肿瘤组织死亡中具有重要的作用,并且有可能是TACE治疗肝癌的其中一重要机制。但由于肝癌易转移和复发特性,任何单一治疗手段均不满意。选择经肝动脉化疗栓塞术后,但仍有大量病例常存在瘤灶栓塞不充分,TACE时经血管内注入的栓塞剂-碘油化疗药物混合乳剂少,致使瘤灶内碘油化疗药物沉积不充分,无碘油沉积部分病灶则在短期内快速增生,是此类肝癌患者治疗失败和局部复发进展的主要原因。近年,有研究直接将细胞毒药物或生物制剂直接植入肿瘤组织或其周围的间质组织内,对肿瘤细胞进行直接攻击的方法,即经皮穿刺瘤内抗癌药物注射间质治疗(interstitial therapy, IT)。在肿瘤局部缓慢释放,持续保持较高的浓度,杀灭肿瘤细胞,即为缓释化疗(slow-release chemotherapy)。碘油是一种碘含量37%-39%的脂肪酸乙脂亚麻子油,不透X线,具有X线透视下适时监测其弥散范围的优点,同时能选择性地较长时间滞留在肝肿瘤组织中,不像大多数生物载体那样在体内易被降解或失去活性,也不会因癌细胞突变而失去特异性,可以作为较好的载体进行瘤内药物注射。碘油作为粘稠的油性液体,可以完全包围癌细胞阻碍癌细胞与组织液进行物质交换及氧的获取,使细胞水肿和脂肪变性,重度持续缺氧引发细胞凋亡和死亡。目前仅有少量文献报道经皮肝穿刺瘤内注入碘油化疗药物乳剂进行肿瘤间质治疗肝癌的临床研究,其中对肝癌患者免疫功能的影响、诱导肿瘤细胞凋亡的基础研究甚少。我们采用TACE联合经皮肝穿刺瘤内注射碘油化疗药物,进行肿瘤间质治疗,一方面探讨该序贯治疗模式的临床疗效及其病理变化,一方面通过流式细胞仪(FCM)检测外周血T淋巴细胞亚群免疫功能的变化,所诱导的肿瘤细胞凋亡变化,初探其对免疫功能的影响、诱导肿瘤细胞凋亡在该治疗模式中的作用,并预期是其重要的治疗机制之一。我们将该治疗模式的临床研究和基础研究结合在一起,这是本研究的特色创新之处,尚未见类似报道。目的研究单纯经肝动脉化疗栓塞(transcatheter arterial chemoembolizaton,TACE)联合经皮肝瘤内碘油化疗药物注射肿瘤间质治疗(interstitial therapy, IT)肝癌的临床疗效,病理变化,介入术前后肝癌患者外周血T淋巴细胞亚群及免疫球蛋白的变化及意义。探讨联合肿瘤间质治疗后诱导肝癌肿瘤细胞凋亡的变化及其在肿瘤治疗中的作用机制和意义。材料与方法将60例肝癌患者分成两组,对照组(A组)30例行单纯TACE治疗,实验组(B组)30例行TACE联合经皮肝瘤内药物注射碘油化疗药物肿瘤间质治疗。观察患者术后发热等不良反应,术前及术后1周抽取2组患者外周血,常规检测血常规、肝肾功能,观察其变化,术后两月复查和随访,观察2组患者术后一月瘤灶内碘油沉积情况以及术后半年肿瘤局控率,1、2年生存率以及中位生存期。应用流式细胞术检测外周血T淋巴细胞亚群(CD3+T、CD4+T、CDS+T、CD4+T/CD8+T),检测免疫球蛋白和补体C3、C4水平变化,观察免疫功能的改变。术后一月肿瘤穿刺活检取材,病理染色观察病理改变：采用流式细胞仪Annexin-V/PI双染法检测,观察肿瘤细胞凋亡率、死亡率。结果A组患者术后体温正常者7例,低度发热者3例、中度发热7例、高度发热13例,B组体温正常16例、低度发热8例、中度发热4例、高度发热2例,χ2检验两组比较有显著性统计学差异,P<0.01。配对t检验显示B组白细胞、血红蛋白术前术后差值明显低于A组,两组比较有显著性统计学差异,P<0.01,B组血小板术前术后差值低于A组,有统计学差异,P<0.05。术后A组白细胞明显高于B组,两组比较有显著性统计学差异,P<0.01；术后A组血小板、血红蛋白要低于术后B组,但两组比较无统计学意义,P>0.05。配对t检验显示B组谷丙转氨酶、谷草转氨酶术前术后差值明显低于A组,两组比较有显著性统计学差异,P<0.01,B组总胆红素指数术前术后差值低于A组,有统计学差异,P<0.05,B组血白蛋白术前术后差值低于A组,但两组间比较无统计学差异,P>0.05。术后A组谷丙转氨酶、谷草转氨酶、总胆红素指数均明显高于B组、血白蛋白明显低于B组,两组比较有显著统计学差异,P<0.05。配对t检验显示B组血尿素氮术前术后差值低于A组,两组比较有统计学差异,P<0.05；B组血肌苷术前术后差值低于A组,但无统计学意义,P>0.05。术后尿素氮、肌苷A组均高于B组,但两组比较无统计学意义,P>0.05。A组手术后碘油沉积率于<50%、50-75%、>75%范围者各为8、12、10例；B组分别为0、12、8例,A、B两组手术后碘油沉积率比较有显著性统计学差异,P<0.05；A组手术后甲胎球蛋白降低率于<50%、>50%范围者各为13、17例,B组分别为5、25例,A、B两组甲胎球蛋白比较有统计学差异,P<0.05(见表5)。A组手术后肿瘤缩小率于<25%、25%-50%、50-75%、>75%者各为23、6、1、0例；B组分别为17、12、1、0,但A、B两组比较无统计学意义,P>0.05。A组手术后6个月复查稳定无复发进展的为12例,复发进展的为18例；B组分别为20、10例,A、B两组手术后6月局控率相比较有统计学差异,P<0.05。A组1,2年生存率80%,43%,中位生存期20.5月。B组1、2年生存率90%,60%,中位生存期22月。外周血T淋巴细胞亚群的结果显示A组单纯经肝动脉化疗栓塞肝癌患者,手术后一周CD3、CD4及CD4/CD8升高,CD8降低,手术前后CD4及CD4/CD8差异有显著性(P<0.05),CD3、及CD8则无统计学差异；B组联合肿瘤间质治疗后,其外周血T淋巴细胞较A组变化更为明显,手术前后CD3、CD4及CD4/CD8升高,CD8降低,手术前后具有非常显著性统计学差异(P<0.01),且A、B两组术后比较,除CD3外统计学亦有显著性差异(P<0.05)。免疫球蛋白和补体检测结果,A组术后免疫球蛋白和补体有所升高,以IgG、IgA、C4升高明显,手术前后有显著性统计学差异,(P<0.05),但IgM、C3手术前后无显著性统计学差异(P>0.05)；B组手术前后仅IgG有所升高,具有显著性统计学差异(P<0.01),IgA、IgM、C3、C4无显著性统计学差异(P>0.05),B组IgG、IgM手术前后差值较A组升高,有显著性统计学差异(P<0.05), IgA、C3、C4无显著性统计学差异(P>0.05)。A组瘤灶病理以大量肿瘤细胞凝固性坏死改变为主,B组则可见大量肿瘤细胞脂肪样变性坏死。A、B两组患者正常肝脏组织流式细胞仪Annexin V/PI双染法凋亡率相比较无统计学意义；两组正常肝脏组织的坏死率比较也无统计学意义；B组患者肿瘤组织凋亡率显著高于A组,两组相比较有统计学差异,P<0.05；A组肿瘤边缘组织死亡率显著高于B组,两组死亡率比较有显著性统计学差异,P<0.01。结论TACE联合肿瘤间质注射碘油化疗药物治疗肝癌,可以显著提高瘤灶碘油沉积率,提高肿瘤局控率,降低TACE术后残留瘤灶复发率,延长患者生存期,减轻介入术后不良反应。单纯TACE治疗肝癌增强患者细胞免疫功能,对体液免疫影响较小。联合瘤内碘油化疗药物乳剂注射肿瘤间质治疗的方法可以更大程度改善患者细胞免疫和体液免疫功能。经动脉化疗栓塞诱导大量癌细胞进入凋亡,是TACE联合瘤内药物注射碘油化疗药物肿瘤间质治疗肝癌的重要机制,该治疗模式可以促进肝癌细胞凋亡,对于进一步提高肝癌的疗效具有积极的意义,而其所引致的急性凝固性坏死改变、大量急性肿瘤溶解坏死等不良反应较少,因此是一种相对安全、稳妥、有效、和具有前景的肝癌治疗模式。更多还原

【Abstract】 BackgroundThe development of primary hepatic carcinoma is a process with multiple factor, multiple stage, polygenic variation, close correlated with immune state, especially T lymphocyte cell. The process is regulated by multiple factor, accompanied with confused immune state to some extent. Majority scholar think that cell mediated immunity occupy the master position in immune. Some research showed the proportion and metergasis of T lymphocyte cells and NK cells are close related with the development and diffusion of tumor. And some research showed that of T lymphocyte subsets in peripheral blood in patients with HCC, CD3 and CD4 degraded, CD8 increased, immune function was weak, that was the major cause of weak immune of which tumor cells resulted in immune suppression.Tumor immunity is related with T lymphocyte cell mediated immunity in major, which reflect organism immune function. T lymphocyte subsets are consist of different subsets, especially CD4/CD8. It is the sensitive reflection against the balance of cell mediated immunity. CD4+T lymphocyte cells are the core of the cell mediated immunity, which regulate immunoreactive function by excreting cytokine, named helper T lymphocyte. Th is the restriction T lymphocyte of major histocompatibility antigen(MHC)classⅡmolecule. Because most tumor cells do not present MHCⅡ, this kind molecule do not direct identify tumor cells, but rely on antigen presenting cell, such as B lymphocyte, macrophagus to present related antigen and secret lymphokine, interleukin-2, r-interferon, and so on, so that activate B lymphocyte, macrophagus, NK cell to play its effective resisting tumor.Substantial evidence that has been expressed indicates that the cell apoptosis is related with the development of the tumor, the occurrence of the tumor closely, and hepatoma carcinoma cells cultured in vitro and esoteric all existed phenomenon of apoptosis. Spontaneous malignant cell apoptosis happened to hepatoma untreated, and malignancy to much more extent, higher index of apoptosis. It is positive correlation between proliferation index and apoptotic index in liver cancer. From normal liver to lesion precancerous and hepatoma, apoptosis percentage increased as proliferation increased. It is obviously higher of apoptosis in liver cancer tumor tissue than in the normal liver tissue. There are two sides of apoptois probably, one side restraining growth, another eliminating weak vitality tumor cells so that strong invasiveness tumor cells cloning and developing. So to study the mechanism and effectiveness of apoptosis could supply an important theory basis on new pathway of tumor treatment.Primary hepatic carcinoma is one of the most common malignant tumors in our country. Any single therapeutic tool is not satisfactory with liver cancer for the characteristic of easy metastasis and recurrence. Transcatheter arterial chemoembolizaton, TACE, is the first of chosen treatment methods, but there are still insufficenicy of embolization in many cases which lipiodol chemotherapeutics emulsion deposited in tumor focus is not enough so that rapid hyperplasy in tumor focus without lipiodol deposited. So it is the major cause of treatment failure and recurrence progression in local tumor region to the patients with liver cancer. There were any investigations implanting cytotoxic drug or biological agents directly into tumor tissue or peripheral interstitial substance recently, that is interstitial therapy, IT, forming the slow-release chemotherapy which killed tumor cells in local tumor region by slow released chemotherapy and sustained high concentrate agents.Transcatheter arterial chemoembolizaton, TACE, is the first selection to unable removal hepatoma. After TACE, a large amount of tumor cells were killed, coagulation necrosis in tumor appeared and tumor cell burden alleviate, so immune suppression factors, TNF, decreased and weakened its influence to immune function. But Transcatheter arterial chemoembolizaton include chemo and embolization which make influence to organism immune function complex.Lipiodol is a kind of fatty acid aethyl conjulin with iodine contents about 37%-39%. There is property of lipiodol not penetrated by X-ray, monitoring its diffusion scope at the proper moment through X-ray, being detained long term in tumor tissue, not easy to be missing activity and specificity for tumor cell mutation. So lipiodol is a better carrier adapted to injection into tumor, it can surround tumor cell to hinder material and oxygen exchange, then make cells hydropsia, fatty degeneration and death. But there were only few basic researches about percutaneous chemotherapic agents lipiodol emulsion injection.On another respect, common standpoint of the mechanism of TACE is that local chemoembolization bring up the tumor cells necrosis. But recently it was found that ischemic and oxygen deficiency were the reason of inducing tumor cells apoptosis, immediate early gene expression for several minutes after ischemic and oxygen deficiency. Ischemic and oxygen deficiency could induce more than 80 gene expression including that expression of gene promoting apoptosis increases and expression of gene restraining apoptosis descend. So apoptosis play an important role in tumor tissue necrosis due to TACE in patients with hepatoma. And it is an important mechanism to TACE probably of the apoptosis induced by TACE.We combined TACE with percutaneous chemotherapic agents lipiodol emulsion injection to make interstitial therapy in liver cancer, in order to approach the clinic curative effect of this sequential therapy and pathological change in one side, to explore the characteristics and significances in immunology changes of blood subgroups of lymphocyte T, to explore significances and effective of the tumor cell apoptosis induced by the treatment method in another side. We got the clinic research and basic research together, that is special feature, new ideals in our study, and not yet reported.ObjectiveTo study the clinic curative effect by transcatheter arterial chemoembolizaton combined with interstitial therapy for liver cancer and pathological change first, to explore the characteristics and significances in immunology changes of blood subgroups of lymphocyte T, to explore significances and effective of the tumor cell apoptosis induced by the this sequential therapy secondly.Methods60 patients with liver cancer were divided into two groups, A group received transcatheter arterial chemoembolizaton, and B group received transcatheter arterial chemoembolizaton combined with interstitial therapy of percutaneous lipiodol and anti-cancer agents injection intratumorly for liver cancer. Then to observe the percent of lipiodol deposited in the tumor, local control ratio of 6 months, survive rate in 2 years, the median survival time, adverse effect, and puncher biopsy one month postoperative. T lymphocyte cell subsets, CD3+, CD4+,CD8+, CD4+/CD8+ in the peripheral blood before and one week after operation were measured by flow cytometry; immune globulin determined by single radial immunodiffusion. The apoptosis percentage, death percentage of tumor cells was measured by flow cytometry with Annexin V/PI staining.ResultsThere was significant difference in the percent of fever between two groups, P< 0.01, normal temperature 7 instances in A group, low fever 3 instance, middle lever 7 instances, high lever 13 instances; normal temperature 16 instances in B group, low fever 8 instance, middle lever 4 instances, high lever 2 instances. There was no significant difference of WBC, HB, PLB preoperative in both A and B groups (P> 0.05).There was significant difference of WBC, HB between preoperative and postoperative in both A and B groups (P<0.05). And significant difference of WBC, HB between A and B group postoperative (P<0.05). It was higher of WBC in A group preoperative than postoperative, there was significant difference, P<0.05, but in B group there was no significant difference, P>0.05. And it was higher of HB in A group preoperative than postoperative, there was significant difference, P<0.05, but in B group there was no significant difference, P>0.05. There was significant difference in the percent of lipiodol deposited in the tumor, local control ratio of 6 months, survive rate in 2 years, the median survival time between A and B groups (P <0.05). There was significant difference in the rate of AFP degrade between A and B groups (P<0.05) postoperative. There is 13,17examples respectively in A group the rate of AFP degrade<50%,>50%, and B group 5,25 respectively. And there was a great quantity of coagulation necrosis of tumor focus in A group pathology postoperative, but a great quantity of lipoid degeneration necrosis in B group pathology postoperative. It was higher of CD3, CD4, CD4/8 in postoperative than in preoperative of both A and B groups. There was significant difference of CD3, CD4, CD4/CD8 between preoperative and postoperative in both A and B groups (P<0.05). And significant difference between A and B group postoperative exclude CD8 (P< 0.05). It was higher of immune globulin in postoperative than in preoperative of both A and B groups. But there was no significant difference between postoperative and preoperative of A group (P>0.05); immune globulin exclude C3significant difference between postoperative and preoperative of B group (P<0.05). It was no less in A group than in B group of the apoptosis percentage in normal tissue. There was no significant difference of the apoptosis percentage in normal tissue between A and B group (P>0.05). But it was higher in A group than in B group of the necrosis percentage in tumor edge. There was very significant difference of the necrosis percentage in tumor edge between A and B group (P<0.01). It was higher in B group than in A group of the apoptosis percentage in tumor edge. There was very significant difference of the apoptosis percentage in tumor edge between A and B group(P<0.05).ConclusionsThere was no less influence on humoral-mediated immunity in single TACE than associated with percutaneous chemotherapic agents lipiodol emulsion injection to make interstitial therapy in liver cancer. This sequence method of treatment could improve cell-mediated immunity and humoral immune function to much more extent. Transcatheter arterial chemoembolizaton combined with interstitial therapy of percutaneous lipiodol and anti-cancer agents injection intratumorly for liver cancer may improve the percent of lipiodol deposited in the tumor, local control ratio of 6 months, and to extend life span, to drop recurrence rate of remained focus tumor, to relieve adverse effect postoperative. So inducing the tumor cell apoptosis and improving immune function by TACE associated with interstitial therapy is the one of the major mechanisms treating liver cancer possibly.更多还原