γ-干扰素在肝癌免疫逃逸中的作用与机制的研究
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摘要
目的与背景：
肝细胞癌(hepatocellular carcinoma, HCC)是人类常见的恶性肿瘤之一,在恶性肿瘤发病率排在第五位。由于早期诊断困难和对各种治疗反应差肝细胞癌的5年死亡率仍较高。尽管在肝癌的早期诊断和小肝癌治疗等方面的研究取得了很大的进展,肝癌的治疗手段仍然是有限的。手术切除是常用的根治HCC的方法,但临床确诊的HCC多属中晚期已丧失了手术的机会,HCC术后复发很常见,HCC患者异体肝移植价格昂贵难以推广。HCC对目前常用的化疗药物抵抗,而且肝功能障碍影响化疗药物的代谢和机体的耐受性。射频消融(Radiofrequency ablation therapy)经皮乙醇注射(percutaneous ethanol injection therapy)和动脉化学栓塞法(transarterial chemoembolization, TACE)是目前常用的HCC治疗法,但疗效有限,多属于姑息性治疗。发展新的治疗方法对改善肝癌的治疗极为重要。
生物治疗(biotherapy)是肝癌治疗中的第四大类疗法,与手术、化疗和放疗三大常规疗法相互补充,免疫治疗是最常用的生物疗法。免疫治疗通过主动和被动方法增强、诱导机体产生针对肝癌细胞的免疫反应,以杀伤肿瘤细胞、抑制肿瘤的生长,作为常规疗法的辅助手段杀伤常规治疗后残存的肿瘤细胞,具有预防复发与转移的作用。研究表明肝癌细胞表达肿瘤抗原甲胎蛋白(a-fetoprotein, AFP)和黑色素瘤相关抗原(Melanoma-associated antigens, MAGE)等,它们能够被机体的免疫系统所识别,成为细胞毒T淋巴细胞识别的靶分子。目前用于HCC治疗的免疫疗法包括应用各种肿瘤疫苗的主动免疫治疗、应用细胞因子诱导的杀伤细胞(cytokine induced killer cells, CIK)和使用抗体的导向治疗等。HCC免疫治疗亟待解决的问题是HCC的免疫逃逸。
肝癌发生发展过程中炎症反应-抗炎反应的失衡是导致HCC免疫逃逸的重要机制。炎症在肿瘤发生发展过程中起重要作用已被广泛接受,几乎所有的肿瘤微环境中都存在着促炎因子,它们通过复杂的网络影响肿瘤的发生和机体对肿瘤的免疫反应,从而影响肿瘤的演进。80%的HCC与B型肝炎病毒(hepatitis B virus,HBV)和C型肝炎病毒(hepatitis C virus,HCV)感染有关,在中国HCC和HBV感染引起的肝炎和肝硬化常相互伴随。在机体针对感染的肝炎病毒的免疫反应过程中产生大量的促炎性细胞因子,如肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α).γ-干扰素(Interferon-γ,IFN-γ)和白细胞介素-12(Interleukine-12,IL-12),它们通过不同的机制激活淋巴细胞、巨噬细胞等杀伤感染病毒的肝细胞,同时引起肝细胞的损伤和肝功能障碍。机体内存在着复杂的抗炎反应机制抑制过度的炎症反应对机体的损害。许多细胞因子具有抗炎作用,如白细胞介素-10(Interleukine-10,IL-10)和转化生长因子-β(Transforming growth factor-β,TGF-β)等。炎症反应和抗炎反应的失衡、相互消长引起机体的免疫功能紊乱,导致机体抗肿瘤免疫功能降低,不能清除肿瘤细胞。炎症反应过程中产生的活性氧、NO等能够损伤肝细胞引起细胞发生基因突变形成肿瘤细胞
IFN-γ是一种多功能细胞因子,在机体的抗感染、抗肿瘤免疫反应中发挥重要作用。诱导IFN-γ产生以增强NK细胞活性和Thl型细胞因子的分泌、增强具有抗肿瘤活性的细胞免疫的产生是肿瘤疫苗等免疫疗法发挥抗肿瘤效应的重要机制。在肝炎病毒感染过程中机体产生IFN-γ,但并不足以清除病毒。在慢性肝炎和HCC患者血液中也存在IFN-γ,肿瘤仍然发生和发展,提示在肝癌的发生发展过程中存在IFN-γ的分泌及其调节和功能障碍。大量的研究表明HCC患者血清IFN-γ水平降低是肝癌免疫逃逸的机制之一,但补充IFN-γ,甚至是大剂量IFN-γ并不能逆转免疫逃逸现象,提示IFN-γ在肝细胞癌发生发展过程中的作用是复杂的,肝癌细胞逃逸IFN-γ抗肿瘤的作用仍有待深入研究。
本实验先以肝细胞癌、慢性乙型肝炎病人和健康献血员为研究对象,检测病人血清中IFN-γ等6种细胞因子的水平和免疫细胞亚群、乙型肝炎病毒的基因型并进行统计分析。结合病人的治疗分析接受经皮注射乙醇治疗后患者血清IFN-γ水平的变化与疗效的关系。在临床研究的基础上采用体外培养的人肝癌细胞系SMMC-7721研究了IFN-γ对肝癌细胞肿瘤抗原表达的影响,从分子水平上探讨IFN-γ在肝细胞肝癌免疫逃逸中的作用与机制。
资料与方法：
1.病例及样本的采集：2002年4月～2007年5月本院住院和门诊治疗的54例原发性肝细胞癌患者,其中男42例,女12例,年龄33～73岁,平均年龄58岁,经CT、MRI、AFP和(或)肝组织病理均证实为肝细胞癌,取样前未进行任何治疗。。
2.主要实验方法：ELISA检测血清细胞因子：采用双抗体夹心法,一抗为生物素标记的抗IFN-γ、TNF-α、IL-2、IL-4、IL-10、IL-12P70抗体,根据样品的OD值及曲线的斜率计算出细胞因子的含量。采用碱性磷酸酶抗碱性磷酸酶法(APAAP)检测外周血CD4+和CD8+T淋巴细胞的百分率。银染法检测T淋巴细胞核仁形成区嗜银蛋白,流式细胞仪测定肝细胞癌患者外周血粘附单个核细胞CD83及HLA-DR表达。实时荧光定量PCR检测血清HBV-DNA含量和HBV基因型。四氮唑(MTT)比色法检测人肝癌细胞增殖活性。用全自动生物化学检测仪测定及配套检测试剂盒检测体外培养的人肝癌SMMC-7721细胞培养上清和细胞匀浆中γ-GT、ALT、AST活性。间接免疫荧光检测人肝癌SMMC-7721细胞株MAGE-1表达。采用双抗体夹心ELISA法检测γ-干扰素处理的人肝癌SMMC-7721细胞株AFP表达。应用亚硫酸氢盐修饰后PCR测序法(Bisulfite Sequencing PCR, BSP)检测γ-干扰素处理的人肝癌SMMC-7721细胞株MAGE-1基因启动子甲基化状态的变化,采用免疫沉淀检测γ-干扰素处理的人肝癌SMMC-7721细胞株c-Jun氨基端激酶活性的影响。
3.统计学方法：计量资料以均数±标准差((?)±s)表示,采用t检验,计数资料用χ2检验(或连续性校正法),数据分析采用SPSS Version 12,p<0.05为统计学上有显著差异。
结果：
1.外周血细胞因子谱型的变化：HCC病人血清IFN-γ含量为4.92±2.15pg/ml,低于健康献血员(19.16±2.34 pg/ml)和慢性乙型肝炎病人(11.42±6.86 pg/ml)(P<0.05),IL-2、IL-12P70含量降低,而TNF-α、TGF-β1、IL-4、IL-10含量增高。根据肝癌体积大小对病人分组比较血清IFN-γ水平,发现随着肿瘤体增大,血清IFN-γ水平下降,调节IFN-γ分泌的IL-12P70水平与IFN-γ的变化趋势是一致的,但病人血液中都存在IFN-γ。根据甲胎蛋白的水平将未经治疗的病人病人分成2组,结果提示甲胎蛋白含量高时血清IFN-γ水平下降,调节IFN-γ分泌的IL-12P70水平与IFN-γ的变化趋势是一致的。
2.外周血细胞亚群的变化：与慢性乙型肝炎病人和健康献血员相比HCC病人CD4+T淋巴细胞、CD4+/CD8+比值降低,反映T淋巴细胞增殖活性的AgNORs降低。黏附细胞悬液CD83和HLA-DR表达降低。
3.乙型肝炎病毒基因型与IFN-γ浓度的关系：采用实时定量PCR检测慢性乙肝病毒携带者、慢性乙型肝炎病人和HCC病人感染的乙肝病毒基因型并分组,比较血清IFN-γ含量。结果表明感染C基因型乙肝病毒HCC病人血清IFN-γ含量低于感染B基因型乙肝病毒者的HCC病人,IFN-γ的含量分别为6.69±2.11和4.36±1.08 pg/ml,(P<0.05)。乙型肝炎患者的变化类似,但不同基因型乙型肝炎病毒携带者之间,IFN-γ的含量无差异。
4.经皮乙醇注射(PEI)可导致肝癌细胞坏死从而减少肝癌细胞数,坏死的瘤体形成自身瘤苗可以诱导机体产生抗肝癌免疫反应。PEI治疗后病人肿瘤体积缩小,AFP含量降低而IFN-γ水平增加。
5.采用不同浓度IFN-γ处理人肝细胞肝癌细胞系SMMC-7721,结果表明IFN-γ对SMMC-7721细胞的增殖有抑制作用,浓度为512U/ml时抑制率为19.4±2.7%,培养上清中AFP含量为0.18±0.03ng/ml,细胞匀浆AFP含量为0.73±0.04ng/ml,仅用全培基培养的SMMC-7721培养上清的AFP含量为0.74±0.05 ng/ml,细胞匀浆AFP含量为0.79±0.07 ng/ml,提示在IFN-γ作用下SMMC-7721细胞AFP表达量下降,ALT、AST和γ-GT活性变化无统计学差异。在IFN-Y作用48h之后512U/ml组SMMC-7721细胞MAGE-1表达下降、MAGE-1基因启动子甲基化程度增加、JNK活性减低。
结论
肝癌的免疫逃逸是成功的肝癌免疫治疗必须克服的严重障碍。造成肝癌的免疫逃逸的机制是多方面的,包括慢性病毒性肝炎时体内的慢性炎症环境、肝炎病毒逃逸免疫攻击、抗凋亡效应及肿瘤细胞产生的免疫抑制因子、抗原调变等。本研究通过对肝癌病人和体外培养的人肝癌细胞SMMC-7721的研究,分析探讨IFN-γ在肝癌免疫逃逸中的作用,得出以下结论：
1.肝细胞癌病人细胞免疫功能低下,存在Th1向Th2免疫漂移现象,血清IFN-γ水平降低,而且肿瘤体积越大IFN-γ水平越低。
2.感染C基因型乙肝病毒的慢性乙型肝炎病人易发生肝癌并且对干扰素反应差,本研究发现感染C基因型乙肝病毒的肝细胞肝癌和慢性乙型肝炎患者血清IFN-γ水平低于感染B基因型乙肝病毒的病人,提示C基因型乙肝病毒可能使被感染的细胞逃逸IFN-γ诱导的抗肿瘤、抗病毒反应,可能是肝细胞癌的发生发展的重要机制。
3.IFN-γ抑制甲胎蛋白的分泌和MAGE-1的表达,MAGE-1的表达的下调部分是由于MAGE-1基因启动子甲基化程度增加实现的,肿瘤细胞免疫原性降低,有助于肝癌细胞免疫逃逸。IFN-γ具有抗增殖和抗肿瘤的作用可能降低肝癌细胞的JNK活性通路。
在肝细胞癌发生发展过程中IFN-γ的分泌及其功能和肿瘤细胞之间的关系可能是互为因果的。乙型肝炎病毒感染引起机体产生针对病毒的免疫反应,IFN-γ分泌增多,抑制肝炎病毒的复制、诱导产生抗肝炎病毒的细胞毒T淋巴细胞杀伤感染的细胞。增多的IFN-γ诱导具有免疫抑制作用和/或生长因子作用的细胞因子产生,这些反应有利于突变的细胞增长；IFN-γ下调甲胎蛋白和MAGE的表达,促进突变的细胞发生抗原调变,逃逸机体的免疫监视功能。而肿瘤细胞分泌的免疫抑制因子进一步地改变细胞因子网络平衡。确切的机制有待深入研究。
Purpose and Background
Hepatocellular carcinoma is one of the most common primary malignant tumors in the human being. Its incidence takes the fifth place among all kinds of malignant tumors. Due to the difficulty in its early diagnosis and its poor response to the treatment, its mortality of 5-year is very high. In spite of the great achievements in its early diagnosis and treatment of small hepatocellular carcinoma, there are only limited therapy methods available for the disease.Surgical resection to remove a tumor is the most frequently adopted therapy to cure tumor patients. However, most of the clinically diagnosed patients of HCC are cases of middle or late cancer, and they have lost the opportunity to be cured with such an operation, after which the disease tends to recur. Liver transplantation is costly, and consequently, hard to popularize. Moreover, HCC resists the chemotherapeutic drugs that are currently used, and liver dysfunction affects the chemotherapy drug metabolism and the tolerance of the organism. Radiofrequency ablation therapy, percutaneous ethanol injection therapy, and transarterial chemoembolization are other therapies for HCC, but they are not effective—they are more or less palliative treatments. Therefore, it is of great significance to develop a new therapy and to improve the treatment for HCC.
Biotherapy is one of the four categories of the HCC therapies. It and the other three conventional therapies, surgical resection, chemotherapy and radiotherapy, complement each other. The biotherapy in common use is immunotherapy. Immunotherapy helps and induces in the organism an immune response against HCC cells so that the tumor cells can be inhibited or killed. As a supplementary means to the conventional therapies, biotherapy can be used to kill the residual tumor cells after conventional treatments. It can prevent the tumor recurrence and metastasis. Studies show that HCC expresses such proteins as a-fetoprotein (AFP) and Melanoma-associated antigens (MAGE). It can be identified by the immune system, and become the target molecules for the cytotoxic T lymphocyte. The immunotherapy for HCC includes a variety of active immune therapy by vaccination against tumor, cytokine-induced killer cells (CIK), and antibody directed therapy, etc. In immunotherapy, the problem demanding urgent solution is immunologic escape.
In liver tumorigenesis and growth, the imbalance between inflammatory reaction and anti-inflammatory reaction is the important mechanism that causes immune escape of HCC. It had been widely accepted that inflammation plays an important part in the development of tumor, for almost in all tumor microenvironments there are pro-inflammatory cytokines. They affect tumorigenesis and the organism's immune response to the tumor with complicated networks, and thus influence the tumor evolution.80%of HCC cases are related to the infection of hepatitis B virus (HBV) or/and hepatitis C virus (HCV). In China, HCC are usually associated with hepatitis and cirrhosis caused with HBV infection. During its immune response to the hepatitis virus infection, the organism generates a large number of pro-inflammatory cytokines such as tumor necrosis factor a (TNF-α), interferon-γ(IFN-γ) and interleukin 12 (IL-12). They activate lymphocytes and macrophages etc. through different mechanisms to destroy the liver cells infected by the virus, and meanwhile, they cause liver cell injury and liver dysfunction. Many cytokines, interleukin 10 (IL-10) and transforming growth factorβ(TGF-β) for example are anti-inflammatory. The imbalance between inflammatory reaction and anti-inflammatory reaction and interdependence induce immune dysfunction and cause the decrease in the immune function against the tumor. As a result the tumor cells cannot be cleared. The substances resulted from inflammation, like reactive oxygen species and NO, cause hepatocellular injury and gene mutation. Consequently, tumor cells are formed.
IFN-γis a multifunctional cytokine, which plays an important part in the immune response of anti-infection and anti-tumor. Inducing IFN-y to improve the NK cell activity, the secretion of Thl type cytokines, and the generation of cellular immunity with anti-tumor activity is fundamental mechanism for the immunotherapy like vaccination against tumor to exert its anti-tumor effect. In the hepatitis virus infection, the organism generates IFN-γ, but inadequate to clear the virus. Patients with chronic hepatitis and HCC have IFN-y in their blood, yet tumor can still development and progression in their body, which means that they have dysfunction in secretion and regulation of IFN-γ. Studies revealed the low serum level of IFN-γis one of the mechanisms of the immune escape in liver cancer. But that fact that the immune escape cannot be reversed by adding IFN-γeven in large does hows that the function of IFN-γin the development and progression of liver tumor is complicated. HCC immune escape from the anti-tumor effect of IFN-γis still a problem to be conquered.
The object of this study is patients with hepatocellular carcinoma or chronic hepatitis B、sound blood donors. The levels of IFN-y, other 6 kinds of cytokines and immune cell subsets, and the genotypes of the hepatitis B virus in the patients'blood serum are measured and compared with those of the sound blood donors. In combination with the treatment, the relationship between therapeutic effectiveness and changes in the serum level of IFN-y after percutaneous ethanol injection is analyzed. On the basis of clinical research, the effect of IFN-γon HCC antigen expression is studied with cultured heptatoma cell line of SMMC-7721. The study on the function and mechanism of IFN-y in the HCC immune escape is made on the molecule level.
Material and Methods
1. Collection of cases and samples
The 54 cases are patients who were either in the in-patient department or hospitalized or in the out-patient department from April 2002 to May 2007.42 of them are male,12 female. They are aged from 33 to 73, with 58 as the average age. CT, MRI, AFP and/or liver pathology all confirm that they were HCC patients. They had not been treated before.
2. Main experimental methods
Double antibody sandwich ELISA method is used. Primary antibody is biotin-labeled antibody which resists IFN-γ, TNF-α, IL-2, IL-4, IL-10, and IL-12P70. The cytokine content is calculated according to according to the OD value of the sample, and the slope of the curve. The percentage of the lymphocytes of CD4+and CD8+T in the peripheral blood is measured with APAAP method. Argentation is adopted to measure the amount of argyrophilia protein in the nucleolus organizer of the T lymphocytes. The flow cytometry is employed to detect the expression of mononuclear cells of CD83 and HLA-DR adherent to culture dish of the HCC patients'peripheral blood. Real time PCR FQ was used to measure the HBV-DNA content in the serum and the HBV genotypes. MTT colormetry reveals the proliferative activity in the human hepatoma cells. Automatic biochemical detector together with the test reagent kits were used to detect the activity ofγ-GT, ALT, and AST in the lysate of the cultured human HCC cells SMMC-7721. Double antibody sandwich ELISA method is applied to detect the AFP expression of IFN-γ-treated SMMC-7721 cell line. indirect immunofluorescence method is applied to detect the expression of MAGE-1 in SMMC-7721 treated by IFN-γ. bisulfite-sequencing PCR is used to assay the methylation status of MAGE-1 gene promoter of SMMC-7721 treated by IFN-γ.
Immunoprecipitation is used to assay the activity of the c-Jun N-terminal kinases in the HCC cell line of SMMC-7721 treated by IFN-y.
3. Statistical Methods
The measurement data is expressed through mean±standard deviation (x±s), and t test; the enumeration data is checked withχ2 (or method of continuity correction). The data is analyzed with SPSS Version 12, with p< 0.05 as statistically significant difference.
Results
1. Changes in cytokine type of the peripheral blood
IFN-γcontent in the serum of HCC patients is 4.92±2.15pg/ml, lower than that of the sound blood donors (19.16±2.34 pg/ml) and that of the patients who have chronic hepatitis B (11.42±6.86 pg/ml) (P< 0.05, and IL-2 and IL-12P70 content is also lower, but TNF-α, TGF-β1, IL-4 and IL-10 content is higher. The patients are divided into groups according to the size of the tumors. Comparison shows that the bigger tumor a patient has, the lower his IFN-γlevel is. And the changes in their regulation of IFN-γsecretion follow the same pattern. IFN-γ, however, can be found in every patient's blood. The untreated patients are divided into 2 groups according to the level ofα-fetoprotein. The comparative study shows that the IFN-γlevel declines as theα-fetoprotein content rises, but that the IL-12P70 level which regulates the IFN-y secretion has the same tendency with the IFN-γlevel.
2. Changes in subsets of the peripheral blood mononuclear cells The percent of CD4+peripheral blood T lymphocytes, CD83+and HLA-DR+in HCC patients are lower than that of the chronic hepatitis B patients and the blood donors. AgNORs which represent the proliferative activity of the T lymphocytes was decreased in patients with HCC.
3. The relation between the genotypes of hepatitis B virus and IFN-y concentration
The real-time PCR is taken to classify the genotypes of the hepatitis B virus in the chronic hepatitis B virus carriers, chronic hepatitis B patients and HCC patients, and compare the IFN-γcontent in the serum. The result is that the IFN-γcontent in the serum of HCC patients who have been infected with the hepatitis B virus of genotype C is lower than that of the patients who have been infected with the hepatitis B virus of genotype B. The former is 6.69±2.11 pg/ml, and the latter 4.36±1.08pg/ml (P<0.05), The changes in hepatitis B patients are similar, but carriers of different hepatitis B virus genotypes have no difference in the IFN-γcontent.
4. Percutaneous ethanol injection (PEI) can induce the hepatoma cells necrosis, and thus can reduce the number of the tumor cells and self-tumor vaccines help the organism generate immune responses against hepatocellular carcinoma. After PEI treatment, the tumor shrinks, AFP level dropped, and IFN-γlevel rised.
5. IFN-γof different concentrations is applied to culture the human HCC cell line SMMC-7721. The concentration ofα-fetoprotein in the cell lysate of the cultured SMCC-7721 was measured with ELISA method. The result is that IFN-γcan obviously inhibit the proliferation of SMMC-7721, that the inhibition ratio is 19.4±2.7%at the concentration of 512U/ml, that the concentration ofα-fetoprotein in the medium of SMMC-7721 cultured with IFN-γwas 0.18±0.03ng/ml, and that in the lysate was 0.73±0.04ng/ml. The concentration ofα-fetoprotein in the medium of SMMC-7721 cultured with complete medium was 0.74±0.05 ng/ml, and that in the lysate was 0.79±0.07 ng/ml. The above data showed that the expression ofα-fetoprotein in SMMC-7721 declined under the action of IFN-γ. that the changes in the activity of ALT, AST andγ-GT is not significant. the methylation status of MAGE-1 gene promoter of SMMC-7721 was increased and the activity of JNK was decreased treated by IFN-y. Discussion and Conclusion
The immunologic escape of liver cancer is the serious barrier to break down in immunotherapy. It is caused by different mechanisms, including the chronic inflammatory environment of the body in the period of chronic virus hepatitis, the immune escape of liver cancer, anti-apoptotic effect, the immunosuppressive factor and antigenic modulation of the tumor cells. Based on research made in patients with HCC and the cultured human hepatoma cells of SMMC-7721, role of IFN-γin the immune escape of liver cancer was discussed. Main conclusions were followed:
1. In HCC patients, the immune function is decreased, there was the immune shift from Thl to Th2, the IFN-γlevel in the serum is low than that of patients with chronic hepatitis and sound blood-donors, and the IFN-γlevel decreased as the size of the tumor increased.
2. The patients with chronic hepatitis B virus of genotype C are more likely to develpe HCC, and they react poorly to interferon. The IFN-γlevel in their serum is discovered to be lower than that that in the patients with chronic hepatitis B virus of genotype B. That means that hepatitis B virus of genotype C may allow the infected cells to escape the antitumor and antivirus reactions induced by IFN-γ. That is one of pathogenesis of development and progression of HCC.
3. IFN-y inhibits the secretion ofα-fetoprotein and the expression of MAGE-1, which contributed to the reduction of the tumor cell immunogenicity and the immune escape of liver cancer. IFN-y down-regulated the expression of MAGE-1, realized methylation status of MAGE-1 gene promoter partily, and habinted cellular proliferation of SMMC-7721 through JNK route.
In the process of tumorigenesis and growth of HCC, there is reciprocal relationship between the tumor cells and IFN-γ. Hepatitis B virus caused immune response to the virus, and the secretion of IFN-γincreased so that the replication of the virus is inhibited. As a result, the cytotoxic T lymphocytes were activated and proliferated to kill the infected cells. The increased IFN-y promoted the generation of cytokines with immunosupressive and growth factor activity. Such reactions were helpful to the growth of mutant cells. IFN-y down-regulated the expression ofα-fetoprotein and MAGE-1, and promoted the antigenic modulation of the mutant cells leading to the cancer cells to escape the immune surveillance in the organism. The immunosuppressive factor secreted by the tumor cells further changed the balance of the cytokine network. But the exact mechanism is still to be investigated.
肝细胞癌(hepatocellular carcinoma, HCC)是人类常见的恶性肿瘤之一,在恶性肿瘤发病率排在第五位。由于早期诊断困难和对各种治疗反应差肝细胞癌的5年死亡率仍较高。尽管在肝癌的早期诊断和小肝癌治疗等方面的研究取得了很大的进展,肝癌的治疗手段仍然是有限的。手术切除是常用的根治HCC的方法,但临床确诊的HCC多属中晚期已丧失了手术的机会,HCC术后复发很常见,HCC患者异体肝移植价格昂贵难以推广。HCC对目前常用的化疗药物抵抗,而且肝功能障碍影响化疗药物的代谢和机体的耐受性。射频消融(Radiofrequency ablation therapy)经皮乙醇注射(percutaneous ethanol injection therapy)和动脉化学栓塞法(transarterial chemoembolization, TACE)是目前常用的HCC治疗法,但疗效有限,多属于姑息性治疗。发展新的治疗方法对改善肝癌的治疗极为重要。
生物治疗(biotherapy)是肝癌治疗中的第四大类疗法,与手术、化疗和放疗三大常规疗法相互补充,免疫治疗是最常用的生物疗法。免疫治疗通过主动和被动方法增强、诱导机体产生针对肝癌细胞的免疫反应,以杀伤肿瘤细胞、抑制肿瘤的生长,作为常规疗法的辅助手段杀伤常规治疗后残存的肿瘤细胞,具有预防复发与转移的作用。研究表明肝癌细胞表达肿瘤抗原甲胎蛋白(a-fetoprotein, AFP)和黑色素瘤相关抗原(Melanoma-associated antigens, MAGE)等,它们能够被机体的免疫系统所识别,成为细胞毒T淋巴细胞识别的靶分子。目前用于HCC治疗的免疫疗法包括应用各种肿瘤疫苗的主动免疫治疗、应用细胞因子诱导的杀伤细胞(cytokine induced killer cells, CIK)和使用抗体的导向治疗等。HCC免疫治疗亟待解决的问题是HCC的免疫逃逸。
肝癌发生发展过程中炎症反应-抗炎反应的失衡是导致HCC免疫逃逸的重要机制。炎症在肿瘤发生发展过程中起重要作用已被广泛接受,几乎所有的肿瘤微环境中都存在着促炎因子,它们通过复杂的网络影响肿瘤的发生和机体对肿瘤的免疫反应,从而影响肿瘤的演进。80%的HCC与B型肝炎病毒(hepatitis B virus,HBV)和C型肝炎病毒(hepatitis C virus,HCV)感染有关,在中国HCC和HBV感染引起的肝炎和肝硬化常相互伴随。在机体针对感染的肝炎病毒的免疫反应过程中产生大量的促炎性细胞因子,如肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α).γ-干扰素(Interferon-γ,IFN-γ)和白细胞介素-12(Interleukine-12,IL-12),它们通过不同的机制激活淋巴细胞、巨噬细胞等杀伤感染病毒的肝细胞,同时引起肝细胞的损伤和肝功能障碍。机体内存在着复杂的抗炎反应机制抑制过度的炎症反应对机体的损害。许多细胞因子具有抗炎作用,如白细胞介素-10(Interleukine-10,IL-10)和转化生长因子-β(Transforming growth factor-β,TGF-β)等。炎症反应和抗炎反应的失衡、相互消长引起机体的免疫功能紊乱,导致机体抗肿瘤免疫功能降低,不能清除肿瘤细胞。炎症反应过程中产生的活性氧、NO等能够损伤肝细胞引起细胞发生基因突变形成肿瘤细胞
IFN-γ是一种多功能细胞因子,在机体的抗感染、抗肿瘤免疫反应中发挥重要作用。诱导IFN-γ产生以增强NK细胞活性和Thl型细胞因子的分泌、增强具有抗肿瘤活性的细胞免疫的产生是肿瘤疫苗等免疫疗法发挥抗肿瘤效应的重要机制。在肝炎病毒感染过程中机体产生IFN-γ,但并不足以清除病毒。在慢性肝炎和HCC患者血液中也存在IFN-γ,肿瘤仍然发生和发展,提示在肝癌的发生发展过程中存在IFN-γ的分泌及其调节和功能障碍。大量的研究表明HCC患者血清IFN-γ水平降低是肝癌免疫逃逸的机制之一,但补充IFN-γ,甚至是大剂量IFN-γ并不能逆转免疫逃逸现象,提示IFN-γ在肝细胞癌发生发展过程中的作用是复杂的,肝癌细胞逃逸IFN-γ抗肿瘤的作用仍有待深入研究。
本实验先以肝细胞癌、慢性乙型肝炎病人和健康献血员为研究对象,检测病人血清中IFN-γ等6种细胞因子的水平和免疫细胞亚群、乙型肝炎病毒的基因型并进行统计分析。结合病人的治疗分析接受经皮注射乙醇治疗后患者血清IFN-γ水平的变化与疗效的关系。在临床研究的基础上采用体外培养的人肝癌细胞系SMMC-7721研究了IFN-γ对肝癌细胞肿瘤抗原表达的影响,从分子水平上探讨IFN-γ在肝细胞肝癌免疫逃逸中的作用与机制。
资料与方法：
1.病例及样本的采集：2002年4月～2007年5月本院住院和门诊治疗的54例原发性肝细胞癌患者,其中男42例,女12例,年龄33～73岁,平均年龄58岁,经CT、MRI、AFP和(或)肝组织病理均证实为肝细胞癌,取样前未进行任何治疗。。
2.主要实验方法：ELISA检测血清细胞因子：采用双抗体夹心法,一抗为生物素标记的抗IFN-γ、TNF-α、IL-2、IL-4、IL-10、IL-12P70抗体,根据样品的OD值及曲线的斜率计算出细胞因子的含量。采用碱性磷酸酶抗碱性磷酸酶法(APAAP)检测外周血CD4+和CD8+T淋巴细胞的百分率。银染法检测T淋巴细胞核仁形成区嗜银蛋白,流式细胞仪测定肝细胞癌患者外周血粘附单个核细胞CD83及HLA-DR表达。实时荧光定量PCR检测血清HBV-DNA含量和HBV基因型。四氮唑(MTT)比色法检测人肝癌细胞增殖活性。用全自动生物化学检测仪测定及配套检测试剂盒检测体外培养的人肝癌SMMC-7721细胞培养上清和细胞匀浆中γ-GT、ALT、AST活性。间接免疫荧光检测人肝癌SMMC-7721细胞株MAGE-1表达。采用双抗体夹心ELISA法检测γ-干扰素处理的人肝癌SMMC-7721细胞株AFP表达。应用亚硫酸氢盐修饰后PCR测序法(Bisulfite Sequencing PCR, BSP)检测γ-干扰素处理的人肝癌SMMC-7721细胞株MAGE-1基因启动子甲基化状态的变化,采用免疫沉淀检测γ-干扰素处理的人肝癌SMMC-7721细胞株c-Jun氨基端激酶活性的影响。
3.统计学方法：计量资料以均数±标准差((?)±s)表示,采用t检验,计数资料用χ2检验(或连续性校正法),数据分析采用SPSS Version 12,p<0.05为统计学上有显著差异。
结果：
1.外周血细胞因子谱型的变化：HCC病人血清IFN-γ含量为4.92±2.15pg/ml,低于健康献血员(19.16±2.34 pg/ml)和慢性乙型肝炎病人(11.42±6.86 pg/ml)(P<0.05),IL-2、IL-12P70含量降低,而TNF-α、TGF-β1、IL-4、IL-10含量增高。根据肝癌体积大小对病人分组比较血清IFN-γ水平,发现随着肿瘤体增大,血清IFN-γ水平下降,调节IFN-γ分泌的IL-12P70水平与IFN-γ的变化趋势是一致的,但病人血液中都存在IFN-γ。根据甲胎蛋白的水平将未经治疗的病人病人分成2组,结果提示甲胎蛋白含量高时血清IFN-γ水平下降,调节IFN-γ分泌的IL-12P70水平与IFN-γ的变化趋势是一致的。
2.外周血细胞亚群的变化：与慢性乙型肝炎病人和健康献血员相比HCC病人CD4+T淋巴细胞、CD4+/CD8+比值降低,反映T淋巴细胞增殖活性的AgNORs降低。黏附细胞悬液CD83和HLA-DR表达降低。
3.乙型肝炎病毒基因型与IFN-γ浓度的关系：采用实时定量PCR检测慢性乙肝病毒携带者、慢性乙型肝炎病人和HCC病人感染的乙肝病毒基因型并分组,比较血清IFN-γ含量。结果表明感染C基因型乙肝病毒HCC病人血清IFN-γ含量低于感染B基因型乙肝病毒者的HCC病人,IFN-γ的含量分别为6.69±2.11和4.36±1.08 pg/ml,(P<0.05)。乙型肝炎患者的变化类似,但不同基因型乙型肝炎病毒携带者之间,IFN-γ的含量无差异。
4.经皮乙醇注射(PEI)可导致肝癌细胞坏死从而减少肝癌细胞数,坏死的瘤体形成自身瘤苗可以诱导机体产生抗肝癌免疫反应。PEI治疗后病人肿瘤体积缩小,AFP含量降低而IFN-γ水平增加。
5.采用不同浓度IFN-γ处理人肝细胞肝癌细胞系SMMC-7721,结果表明IFN-γ对SMMC-7721细胞的增殖有抑制作用,浓度为512U/ml时抑制率为19.4±2.7%,培养上清中AFP含量为0.18±0.03ng/ml,细胞匀浆AFP含量为0.73±0.04ng/ml,仅用全培基培养的SMMC-7721培养上清的AFP含量为0.74±0.05 ng/ml,细胞匀浆AFP含量为0.79±0.07 ng/ml,提示在IFN-γ作用下SMMC-7721细胞AFP表达量下降,ALT、AST和γ-GT活性变化无统计学差异。在IFN-Y作用48h之后512U/ml组SMMC-7721细胞MAGE-1表达下降、MAGE-1基因启动子甲基化程度增加、JNK活性减低。
结论
肝癌的免疫逃逸是成功的肝癌免疫治疗必须克服的严重障碍。造成肝癌的免疫逃逸的机制是多方面的,包括慢性病毒性肝炎时体内的慢性炎症环境、肝炎病毒逃逸免疫攻击、抗凋亡效应及肿瘤细胞产生的免疫抑制因子、抗原调变等。本研究通过对肝癌病人和体外培养的人肝癌细胞SMMC-7721的研究,分析探讨IFN-γ在肝癌免疫逃逸中的作用,得出以下结论：
1.肝细胞癌病人细胞免疫功能低下,存在Th1向Th2免疫漂移现象,血清IFN-γ水平降低,而且肿瘤体积越大IFN-γ水平越低。
2.感染C基因型乙肝病毒的慢性乙型肝炎病人易发生肝癌并且对干扰素反应差,本研究发现感染C基因型乙肝病毒的肝细胞肝癌和慢性乙型肝炎患者血清IFN-γ水平低于感染B基因型乙肝病毒的病人,提示C基因型乙肝病毒可能使被感染的细胞逃逸IFN-γ诱导的抗肿瘤、抗病毒反应,可能是肝细胞癌的发生发展的重要机制。
3.IFN-γ抑制甲胎蛋白的分泌和MAGE-1的表达,MAGE-1的表达的下调部分是由于MAGE-1基因启动子甲基化程度增加实现的,肿瘤细胞免疫原性降低,有助于肝癌细胞免疫逃逸。IFN-γ具有抗增殖和抗肿瘤的作用可能降低肝癌细胞的JNK活性通路。
在肝细胞癌发生发展过程中IFN-γ的分泌及其功能和肿瘤细胞之间的关系可能是互为因果的。乙型肝炎病毒感染引起机体产生针对病毒的免疫反应,IFN-γ分泌增多,抑制肝炎病毒的复制、诱导产生抗肝炎病毒的细胞毒T淋巴细胞杀伤感染的细胞。增多的IFN-γ诱导具有免疫抑制作用和/或生长因子作用的细胞因子产生,这些反应有利于突变的细胞增长；IFN-γ下调甲胎蛋白和MAGE的表达,促进突变的细胞发生抗原调变,逃逸机体的免疫监视功能。而肿瘤细胞分泌的免疫抑制因子进一步地改变细胞因子网络平衡。确切的机制有待深入研究。
Purpose and Background
Hepatocellular carcinoma is one of the most common primary malignant tumors in the human being. Its incidence takes the fifth place among all kinds of malignant tumors. Due to the difficulty in its early diagnosis and its poor response to the treatment, its mortality of 5-year is very high. In spite of the great achievements in its early diagnosis and treatment of small hepatocellular carcinoma, there are only limited therapy methods available for the disease.Surgical resection to remove a tumor is the most frequently adopted therapy to cure tumor patients. However, most of the clinically diagnosed patients of HCC are cases of middle or late cancer, and they have lost the opportunity to be cured with such an operation, after which the disease tends to recur. Liver transplantation is costly, and consequently, hard to popularize. Moreover, HCC resists the chemotherapeutic drugs that are currently used, and liver dysfunction affects the chemotherapy drug metabolism and the tolerance of the organism. Radiofrequency ablation therapy, percutaneous ethanol injection therapy, and transarterial chemoembolization are other therapies for HCC, but they are not effective—they are more or less palliative treatments. Therefore, it is of great significance to develop a new therapy and to improve the treatment for HCC.
Biotherapy is one of the four categories of the HCC therapies. It and the other three conventional therapies, surgical resection, chemotherapy and radiotherapy, complement each other. The biotherapy in common use is immunotherapy. Immunotherapy helps and induces in the organism an immune response against HCC cells so that the tumor cells can be inhibited or killed. As a supplementary means to the conventional therapies, biotherapy can be used to kill the residual tumor cells after conventional treatments. It can prevent the tumor recurrence and metastasis. Studies show that HCC expresses such proteins as a-fetoprotein (AFP) and Melanoma-associated antigens (MAGE). It can be identified by the immune system, and become the target molecules for the cytotoxic T lymphocyte. The immunotherapy for HCC includes a variety of active immune therapy by vaccination against tumor, cytokine-induced killer cells (CIK), and antibody directed therapy, etc. In immunotherapy, the problem demanding urgent solution is immunologic escape.
In liver tumorigenesis and growth, the imbalance between inflammatory reaction and anti-inflammatory reaction is the important mechanism that causes immune escape of HCC. It had been widely accepted that inflammation plays an important part in the development of tumor, for almost in all tumor microenvironments there are pro-inflammatory cytokines. They affect tumorigenesis and the organism's immune response to the tumor with complicated networks, and thus influence the tumor evolution.80%of HCC cases are related to the infection of hepatitis B virus (HBV) or/and hepatitis C virus (HCV). In China, HCC are usually associated with hepatitis and cirrhosis caused with HBV infection. During its immune response to the hepatitis virus infection, the organism generates a large number of pro-inflammatory cytokines such as tumor necrosis factor a (TNF-α), interferon-γ(IFN-γ) and interleukin 12 (IL-12). They activate lymphocytes and macrophages etc. through different mechanisms to destroy the liver cells infected by the virus, and meanwhile, they cause liver cell injury and liver dysfunction. Many cytokines, interleukin 10 (IL-10) and transforming growth factorβ(TGF-β) for example are anti-inflammatory. The imbalance between inflammatory reaction and anti-inflammatory reaction and interdependence induce immune dysfunction and cause the decrease in the immune function against the tumor. As a result the tumor cells cannot be cleared. The substances resulted from inflammation, like reactive oxygen species and NO, cause hepatocellular injury and gene mutation. Consequently, tumor cells are formed.
IFN-γis a multifunctional cytokine, which plays an important part in the immune response of anti-infection and anti-tumor. Inducing IFN-y to improve the NK cell activity, the secretion of Thl type cytokines, and the generation of cellular immunity with anti-tumor activity is fundamental mechanism for the immunotherapy like vaccination against tumor to exert its anti-tumor effect. In the hepatitis virus infection, the organism generates IFN-γ, but inadequate to clear the virus. Patients with chronic hepatitis and HCC have IFN-y in their blood, yet tumor can still development and progression in their body, which means that they have dysfunction in secretion and regulation of IFN-γ. Studies revealed the low serum level of IFN-γis one of the mechanisms of the immune escape in liver cancer. But that fact that the immune escape cannot be reversed by adding IFN-γeven in large does hows that the function of IFN-γin the development and progression of liver tumor is complicated. HCC immune escape from the anti-tumor effect of IFN-γis still a problem to be conquered.
The object of this study is patients with hepatocellular carcinoma or chronic hepatitis B、sound blood donors. The levels of IFN-y, other 6 kinds of cytokines and immune cell subsets, and the genotypes of the hepatitis B virus in the patients'blood serum are measured and compared with those of the sound blood donors. In combination with the treatment, the relationship between therapeutic effectiveness and changes in the serum level of IFN-y after percutaneous ethanol injection is analyzed. On the basis of clinical research, the effect of IFN-γon HCC antigen expression is studied with cultured heptatoma cell line of SMMC-7721. The study on the function and mechanism of IFN-y in the HCC immune escape is made on the molecule level.
Material and Methods
1. Collection of cases and samples
The 54 cases are patients who were either in the in-patient department or hospitalized or in the out-patient department from April 2002 to May 2007.42 of them are male,12 female. They are aged from 33 to 73, with 58 as the average age. CT, MRI, AFP and/or liver pathology all confirm that they were HCC patients. They had not been treated before.
2. Main experimental methods
Double antibody sandwich ELISA method is used. Primary antibody is biotin-labeled antibody which resists IFN-γ, TNF-α, IL-2, IL-4, IL-10, and IL-12P70. The cytokine content is calculated according to according to the OD value of the sample, and the slope of the curve. The percentage of the lymphocytes of CD4+and CD8+T in the peripheral blood is measured with APAAP method. Argentation is adopted to measure the amount of argyrophilia protein in the nucleolus organizer of the T lymphocytes. The flow cytometry is employed to detect the expression of mononuclear cells of CD83 and HLA-DR adherent to culture dish of the HCC patients'peripheral blood. Real time PCR FQ was used to measure the HBV-DNA content in the serum and the HBV genotypes. MTT colormetry reveals the proliferative activity in the human hepatoma cells. Automatic biochemical detector together with the test reagent kits were used to detect the activity ofγ-GT, ALT, and AST in the lysate of the cultured human HCC cells SMMC-7721. Double antibody sandwich ELISA method is applied to detect the AFP expression of IFN-γ-treated SMMC-7721 cell line. indirect immunofluorescence method is applied to detect the expression of MAGE-1 in SMMC-7721 treated by IFN-γ. bisulfite-sequencing PCR is used to assay the methylation status of MAGE-1 gene promoter of SMMC-7721 treated by IFN-γ.
Immunoprecipitation is used to assay the activity of the c-Jun N-terminal kinases in the HCC cell line of SMMC-7721 treated by IFN-y.
3. Statistical Methods
The measurement data is expressed through mean±standard deviation (x±s), and t test; the enumeration data is checked withχ2 (or method of continuity correction). The data is analyzed with SPSS Version 12, with p< 0.05 as statistically significant difference.
Results
1. Changes in cytokine type of the peripheral blood
IFN-γcontent in the serum of HCC patients is 4.92±2.15pg/ml, lower than that of the sound blood donors (19.16±2.34 pg/ml) and that of the patients who have chronic hepatitis B (11.42±6.86 pg/ml) (P< 0.05, and IL-2 and IL-12P70 content is also lower, but TNF-α, TGF-β1, IL-4 and IL-10 content is higher. The patients are divided into groups according to the size of the tumors. Comparison shows that the bigger tumor a patient has, the lower his IFN-γlevel is. And the changes in their regulation of IFN-γsecretion follow the same pattern. IFN-γ, however, can be found in every patient's blood. The untreated patients are divided into 2 groups according to the level ofα-fetoprotein. The comparative study shows that the IFN-γlevel declines as theα-fetoprotein content rises, but that the IL-12P70 level which regulates the IFN-y secretion has the same tendency with the IFN-γlevel.
2. Changes in subsets of the peripheral blood mononuclear cells The percent of CD4+peripheral blood T lymphocytes, CD83+and HLA-DR+in HCC patients are lower than that of the chronic hepatitis B patients and the blood donors. AgNORs which represent the proliferative activity of the T lymphocytes was decreased in patients with HCC.
3. The relation between the genotypes of hepatitis B virus and IFN-y concentration
The real-time PCR is taken to classify the genotypes of the hepatitis B virus in the chronic hepatitis B virus carriers, chronic hepatitis B patients and HCC patients, and compare the IFN-γcontent in the serum. The result is that the IFN-γcontent in the serum of HCC patients who have been infected with the hepatitis B virus of genotype C is lower than that of the patients who have been infected with the hepatitis B virus of genotype B. The former is 6.69±2.11 pg/ml, and the latter 4.36±1.08pg/ml (P<0.05), The changes in hepatitis B patients are similar, but carriers of different hepatitis B virus genotypes have no difference in the IFN-γcontent.
4. Percutaneous ethanol injection (PEI) can induce the hepatoma cells necrosis, and thus can reduce the number of the tumor cells and self-tumor vaccines help the organism generate immune responses against hepatocellular carcinoma. After PEI treatment, the tumor shrinks, AFP level dropped, and IFN-γlevel rised.
5. IFN-γof different concentrations is applied to culture the human HCC cell line SMMC-7721. The concentration ofα-fetoprotein in the cell lysate of the cultured SMCC-7721 was measured with ELISA method. The result is that IFN-γcan obviously inhibit the proliferation of SMMC-7721, that the inhibition ratio is 19.4±2.7%at the concentration of 512U/ml, that the concentration ofα-fetoprotein in the medium of SMMC-7721 cultured with IFN-γwas 0.18±0.03ng/ml, and that in the lysate was 0.73±0.04ng/ml. The concentration ofα-fetoprotein in the medium of SMMC-7721 cultured with complete medium was 0.74±0.05 ng/ml, and that in the lysate was 0.79±0.07 ng/ml. The above data showed that the expression ofα-fetoprotein in SMMC-7721 declined under the action of IFN-γ. that the changes in the activity of ALT, AST andγ-GT is not significant. the methylation status of MAGE-1 gene promoter of SMMC-7721 was increased and the activity of JNK was decreased treated by IFN-y. Discussion and Conclusion
The immunologic escape of liver cancer is the serious barrier to break down in immunotherapy. It is caused by different mechanisms, including the chronic inflammatory environment of the body in the period of chronic virus hepatitis, the immune escape of liver cancer, anti-apoptotic effect, the immunosuppressive factor and antigenic modulation of the tumor cells. Based on research made in patients with HCC and the cultured human hepatoma cells of SMMC-7721, role of IFN-γin the immune escape of liver cancer was discussed. Main conclusions were followed:
1. In HCC patients, the immune function is decreased, there was the immune shift from Thl to Th2, the IFN-γlevel in the serum is low than that of patients with chronic hepatitis and sound blood-donors, and the IFN-γlevel decreased as the size of the tumor increased.
2. The patients with chronic hepatitis B virus of genotype C are more likely to develpe HCC, and they react poorly to interferon. The IFN-γlevel in their serum is discovered to be lower than that that in the patients with chronic hepatitis B virus of genotype B. That means that hepatitis B virus of genotype C may allow the infected cells to escape the antitumor and antivirus reactions induced by IFN-γ. That is one of pathogenesis of development and progression of HCC.
3. IFN-y inhibits the secretion ofα-fetoprotein and the expression of MAGE-1, which contributed to the reduction of the tumor cell immunogenicity and the immune escape of liver cancer. IFN-y down-regulated the expression of MAGE-1, realized methylation status of MAGE-1 gene promoter partily, and habinted cellular proliferation of SMMC-7721 through JNK route.
In the process of tumorigenesis and growth of HCC, there is reciprocal relationship between the tumor cells and IFN-γ. Hepatitis B virus caused immune response to the virus, and the secretion of IFN-γincreased so that the replication of the virus is inhibited. As a result, the cytotoxic T lymphocytes were activated and proliferated to kill the infected cells. The increased IFN-y promoted the generation of cytokines with immunosupressive and growth factor activity. Such reactions were helpful to the growth of mutant cells. IFN-y down-regulated the expression ofα-fetoprotein and MAGE-1, and promoted the antigenic modulation of the mutant cells leading to the cancer cells to escape the immune surveillance in the organism. The immunosuppressive factor secreted by the tumor cells further changed the balance of the cytokine network. But the exact mechanism is still to be investigated.
引文
1. Lu CY., Changelian, Unanue E. R.Humana-fetoprotein (AFP) causes a selective down regulation of monocyte MHC class Ⅱ molecules without altering other induced or noninduced monocyte markers or functions in monocytoid cell lines.Cell Immunol,1991,133:506..
2. Murgita A., Goidl S. α-Fetoprotein induces suppressor T cells in vitro. Nature, 1977,267:257.Gabrilovich D.Mechanisms and functional significance of tumour-induced dendritic-cell defects[J].Nat Rev Immunol,2004,4(12):941-952.
3. Peck A, Murgita R A., Wigzell H. Cellular and genetic restrictions in the immunoregulatory activity of α-fetoprotein. J Exp Med,1978,147:667-672.
4. Soon HU, Mulhall C, Alisa A, et al. Alpha-fetoprotein impairs APC function and induces their apoptosis.J Immunol,2004,173 (3):1772-1778.
5. Peng JR, Chen HS, Mou DC. Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters. Cancer Lett,.2005,219(2):223-32.
6. Caballero OL, Chen YT. Cancer/testis (CT) antigens:potential targets for immunotherapy.2009,100(11):2014-21.
7. Chen G, Luo D Nov-Dec; Expression of decoy receptor 3 in liver tissue microarrays. Natl Med J India,2008,21(6):275-8.
8. Chen C, Zhang C, Zhuang G..Decoy receptor 3 overexpression and immunologic tolerance in hepatocellular carcinoma (HCC) developme.Cancer Invest,2008 D, 26(10):965-74.
9. Yang C, Hsieh S L, Teng C M, et al. Soluble Decoy Receptor 3 Induces Angiogenesis by Neutralization of TL1A, a Cytokine Belonging to Tumor Necrosis Factor Superfamily and Exhibiting Angiostatic Action.Cancer Res, 2004,64:1122-1129.
10. Yang C R, Hsieh S L, Ho F M. Decoy Receptor 3 increases monocyte adhesion to endothelial cells via NF-κB dependent up-regulation of Intercellular Adhesion Molecule-1, VCAM-1, and IL-8 expression. The Journal of Immunology,2005, 174:1647-1656.
11. Yang XR, Xu Y, Yu B, et al. High expression levels of putative hepatic stem/ progenitor cell biomarkers related to tumour angiogenesis and poor prognosis of
hepatocellular carcinoma. Gut.,2010 May 4. [Epub ahead of print]
12. Sutton A, Friand V, Brule-Donneger S, et al.Stromal cell-derived factor-1/ chemokine (C-X-C motif) ligand 12 stimulates human hepatoma cell growth, migration, and invasion. Mol Cancer Res,2007,5:21-33.
13. Liu H,Pan Z,Li A,et al. Roles of chemokine receptor 4 (CXCR4) and chemokine ligand 12 (CXCL12) in metastasis of hepatocellular carcinoma cells. Cell Mol Immunol.2008,5:373-378.
14. Chu H, Zhou H, Liu Y, et al. Functional expression of CXC chemokine recepter-4 mediates the secretion of matrix metalloproteinases from mouse hepato-carcinoma cell lines with different lymphatic metastasis ability. Int J Biochem Cell Biol,2007,39:197-205.
15. Rubie C, Frick VO, Wagner M, et al.Chemokine expression in hepatocellular carcinoma versus colorectal liver metastases. World J Gastroenterol,2006,12: 6627-6633.
16. Rubie C, Frick VO, Wagner M, et al. Enhanced expression and clinical significance of CC-chemokine MIP-3 alpha in hepatocellular carcinoma.Scand J Immunol,2006,63:468-477.
17. Matsubara T, Ono T, Yamanoi A, et al. Fractalkine-CX3CR1 axis regulates tumor cell cycle and deteriorates prognosis after radical resection for hepatocellular carcinoma. J Surg Oncol,2007,95:241-249.
18. Miihlbauer M, Ringel S, Hartmann A, et al.Lack of association between the functional CX3CR1 polymorphism V249I and hepatocellular carcinoma. Oncol Rep,2005,13:957-963.
19. Nagaoka S, Yoshida T, Akiyoshi J, et al. The ratio of serum placenta growth factor to soluble vascular endothelial growth factor receptor-1 predicts the prognosis of hepatocellular carcinoma. Oncol Rep,2010,23(6):1647-54.
20. Heneghan MA. Frequency and nature of cytokine gene polymorphisms in hepato-cellular carcinoma in Hong Kong Chinese. Int J Gastrointest.Cancer,2003,34: 19-26.
21. Shin HD. Interleukin 10 haplotype associated with increased risk of hepatocellular carcinoma. Hum Mol Genet,2003,12,901-906.
22. Simona Ognjanovic, Jian-Min Yuan, Ann K.Genetic polymorphisms in the cytokine genes and risk of hepatocellular carcinoma in low-risk non-Asians of USA. Carcinogenesis,2009,30(5):758-762.
23. Dong Z Z, Yao D F, Yao M, et al.Clinical impact of plasmaTGF betal and circulatingTGF betal mRNA in diagnosis of hepatocellular carcinoma. Hepatobiliary [J].Pancreat Dis Int,2008,7(3):288 295.
24. Benetti A, Berenzi A, Gambarotti M, et al.Transforming growth factor-betal and CD105 promote the migration of hepatocellular carcinoma-derived endothelium. Cancer Res,2008,15,68(20):8626-34.
25. Chen J, Zheng XH, Tang XP, et al.A comparative study of serum sFas in patients with hepatocellular cancer and chronic hepatitis]. Hunan Yi Ke Da Xue Xue Bao,2001,26:173-174.
26. Rahman N, Hanaa M. Serum levels of soluble Fas, soluble tumor necrosis factor-receptor Ⅱ, interleukin-2 receptor and interleukin-8 as early predictors of hepatocellular carcinoma in Egyptian patients with hepatitis C virus genotype-4. Comp Hepatol,2010,9:1-5.
27. Shi F,Shi M,Zeng Z,et al. PD-1 and PD-L1 upregulation promotes CD8(+)T-cell apoptosis and post-operative recurrence in hepatocellular carcinoma patients.Int J Cancer,2010,4:19-24.
28. NakamotoY, Kaneko S. Dendritic cell-based immunotherapy for hepatocellular carcinoma. Gan To Kagaku Ryoho,2010,37(3):413-6.
29. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance[J]. Nat Rev Cancer,2005,5(4):263-274.
30. Shevach EM, DiPaolo RA, Andersson J, et al.The lifestyle of naturally occurring CD4+CD25+FoxP3+regulatory T cells[J].Immunol Rev,2006,212:60-73.
31. Shevach EM, DiPaolo RA, Andersson J, et al.The lifestyle of naturally occurring CD4+CD25+FoxP3+regulatory T cells [J].Immunol Rev,2006,212:60-73.
32. Enarsson K, Johnsson E, Lindholm C, et al. Differential mechanisms for T lymphocyte recruitmentin normal and neoplastic human gastric mucosa.[J] Clin Immunol,2006,118(1):24-34.
33. Enarsson K, Johnsson E, Lindholm C, et al. Differential mechanisms for T lymphocyte recruitmentin normal and neoplastic human gastric mucosa.[J] Clin Immunol,2006,118(1):24-34.
34. Kobayashi N, Hiraoka N, Yamagaini W, et al.FOXP3+Regulatory T cells affect the development and progression of hepatocarcinogenesis. Clin Cancer Res, 2007,13(3):902-911.
35. Zeng Z, Yao W, Xu X, et al. Hepatocellular carcinoma cells deteriorate the biophysical properties of dendritic cells. Cell Biochem Biophys,2009, 55(1):33-43.
36. Chew V, Tow C, Teo M, et al. Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients.J Hepatol, 2010,52(3):370-9.
37. Nagao M, Nakajima Y, Hisanaga M, et al. The alteration of Fas receptor and ligand system in hepatocellular carcinomas:how do hepatoma cells escape from the host immune surveillance in vivo? Hepatology,1999,30(2):413-21.
38. Dranoff G, Mulligan RC. Gene transfer as cancer therapy. Adv Immunol.1995, 58:417-54.
39. Beatty GL, Paterson Y. IFN-gamma can promote tumor evasion of the immune system in vivo by down-regulating cellular levels of an endogenous tumor antigen[J]. Immunol,2000,165:5502-5508
40. Le Poole IC, Riker AI, Quevedo ME, et al.Interferon-gammar reduces melanosomal antigen expression and recognition of melanoma cells by cytotoxic T. [J]. Am J Pathol,2002,160:521-528
41. He F, Wang XH, Zhang GM, et al. Sustained low-level expression of interferon-γ promotes tumor development:Potential insights in tumor prevention and tumor immunotherapy. Cancer Immunol Immunother,2005,54:891-897.
1. Sean F, Altekruse, Katherine A, el al. Hepatocellular Carcinoma Incidence, Mortality, and Survival Trends in the United States From 1975 to 2005. J Clin Oncol,2009,27(9):1485-1491.
2. Rampone B, Schiavone B, Martino A, el al. Current management strategy of hepatocellular carcinoma.World J Gastroenterol,2009,15(26):3210-3216.
3. Yau T, Chan P, Epstein R, el al. Evolution of systemic therapy of advanced hepatocellular carcinoma.World J Gastroenterol.2008,14(42):6437-6441.
4. Choi D, Lim H, Lee WJ, el al. Radiofrequency Ablation of Liver Cancer:Early Evaluation of Therapeutic Response with Contrast-Enhanced Ultrasonography. Korean J Radiol.2004,5(3):185-198.
5. Lin SM, Lin CJ, Lin CC, el al. Randomised controlled trial comparing percutaneous radiofrequency thermal ablation, percutaneous ethanol injection, and percutaneous acetic acid injection to treat hepatocellular carcinoma of 3 cm or less. Gut,2005,54(8):1151-1156.
6. Llovet JM, Real MI, Montana X, el al. Arterial embolisation or chemo-embolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma:a randomised controlled trial. Lancet,2002,59: 1734-1739.
7. Zhang W, Liu J, Wu Y, el al. Immunotherapy of hepatocellular carcinoma with a vaccine based on xenogeneic homologous a fetoprotein in mice. Biochemical and Biophysical Research Communications.2008,376:10-14.
8. Sarobe P, Feijoo'E, Alfaro C, el al. MAGE antigens:therapeutic targets in hepatocellular carcinoma?. Journal of Hepatology,2004,40(1):155-158.
9. Greten TF, Manns MP, Korangy F. Immunotherapy of hepatocellular carcinoma. Journal of Hepatology,2006,45:868-878.
10. Porta C, Larghi P, Rimoldi M, el al.Cellular and molecular pathways linking inflammation and cancer. Immunobiology,2009,214:761-77.
11. McGlynn KA, London WT. Epidemiology and natural history of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol,2005,19:3-23.
12. Chisari FV, Isogawa M, Wieland SF. Pathogenesis of hepatitis B virus infection. Pathol Biol (Paris),2010. [Epub ahead of print]
13. Nardin A. Abastado JPFront Biosci. Macrophages and cancer,2008,13:3494-505.
14. Okada T, Sada T, Kubota K. Deferoxamine enhances anti-proliferative effect of interferon-γ against hepatocellular carcinoma cells.Cancer Lett,2007,248: 24-31.
15. Kremsdorf D, Soussan P, Paterlini-Brechot P, el al.Hepatitis B virus related hepatocellular carcinoma:paradigms for viral-related human carcinogenesis. Oncogene,2006,25:3823-3833.
16. Tu Z, Bozorgzadeh A, Pierce RH, el al. TLR-dependent cross talk between human Kupffer cells and NK cells. J Exp Med,2008,205:233-244.
17. Chen Y M, Yang W K, Whany P J, et al. Restoration of the immunocompetence by IL-2 activation and TCR-CD3 engagement of the in vivo anergized tumor-specific CTL from lung cancer patients [J]. Immunother,1997,20:354-358.
18. Sal B, Marotta A, Matthewson C, el al. Investigation of the Mek-MAPkinase-Rsk pathway in human breast cancer. Anticancer Res,1999,19:731-740.
19. Shimizu Y, Iwatsuki S, Herberman R et al. Effects of cytokines on in vitro growth of tumor-infiltrating lymphocytes obtained from human primary and metastatic liver tumors. Cancer Immunol. Immunother,1991,32:280-288.
20. Nakazaki H. Preoperative and postoperative cytokines in patients with cancer[J]. Cancer,1992,70:709.
21. Hus U, Lees B, Kinc M, et al. Papid recruitment of macrophages in interleukin-12-mediated tumor regression. Immunology,1998,95:156-163.
22. Lars A Ormandy, Anatol Farber, Tobias Cantz et al. Direct ex vivo analysis of dendritic cells in patients with hepatocellular carcinoma. World J Gastroenterol. 2006,12(20):3275-3282.
23. Um SH, Mulhall C, Alisa A, et al. Alpha-fetoprotein impairs APC function and induces their apoptosis. J Immunol.2004,173:1772-1778.
24. Nishioka Y, Wen H, Mitani K et al. Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells:a critical role for nitric oxide. LeucocBiol,2003,73:621-629.
25. Lo CH, Chang CM, Tang SW, et al. Differential antitumor effect of interleukin-12 family cytokines on orthotopic hepatocellular carcinoma. Gene Med,2010, 12(5):423-34.
26. Whiteside TL, Herberman RB. The role of natural killer cells in human disease. Clin Immunol Immunopathol,1989,53:1-23.
27. Maione S, Lamberti L. Nucleolar organizer regions activity in lymphocytes of patientswith laryngeal carcinoma. Soc Ital Biol Sper,1993,69:741.
28. Norder H, Courouce AM, Coursaget P, et al.Genetic diversity of hepatitis B virus strains derived worldwide:genotypes, subgenotypes, and HBsAg subtypes. Intervirology,2004,47:289-309.
29. Pujol FH, Navas MC, Hainaut P, et al. Worldwide genetic diversity of HBV genotypes and risk of hepatocellular carcinoma. Cancer Lett,2009,286(1):80-88.
30. Raimondi S, Maisonneuve P, Bruno S, et al.Is response to antiviral treatment influenced by hepatitis B virus genotype?. J Hepatol,2010,52:441-9.
31.Yu MW, Yeh SH, Chen PJ, et al. Hepatitis B virus genotype and DNA leveland hepatocellular carcinoma:a prospective study in men. J Natl Cancer Inst,2005, 97(4):265-72.
32. Kao JH, Chen PJ, Lai MY, et al.Genotypes and clinical phenotypesof hepatitis B virus in patients with chronic hepatitis B virus infection. J Clin Microbiol, 2002,40(4):1207-9.
33. Kao JH, Chen PJ, Lai MY, et al. Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers.J Med Virol,2004,72(3):63-9.
34. Pujol FH, Navas MC, Hainaut P, et al.Worldwide genetic diversity of HBV genotypes and risk of hepatocellular carcinoma. Cancer Lett,2009,286(1):80-8.
35. Ding X, Mzokami M, Yao G, et al. Hepatitis B virus genotype distribution among chronic hepatitis B carriers china shanghaj.[J] Interviology,2001,44(1):43-46.
36. Orito E, Ichida T, Sakugawa H, et al.Geographic distribution ofhepatitis B virus genotype in patients with chronic HBV infection in Japans. [J] Hepatology, 2001,34(3):590-594.
37. Yang HI, Yeh SH, Chen PJ, et al. Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma J. Natl Cancer Inst.2008,100(16):1134-1143.38、Kao JH, Chen PJ, Lai MY, et al. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology,2003,124 (2):327-34.
38. Orito E, Mizokami M, Sakugawa H, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Hepatology,2001,33(1):218-23.
39. Ding X, Mizokami M, Ge X, et al.Different hepatitis B virus genotype distributions among asymptomatic carriers and patients with liver diseases [J] Hepatol Res,2002,22(1):37-44.
40. Gaglio P, Singh S, Degertekin B, et al. Impact of the hepatitis B virus genotype on pre-and post-liver transplantation outcomes. Liver Transpl.,2008,14(10): 1420-7.
41. Huang Y, Wang Z, An S, et al. Role of hepatitis B virus genotypes and quantitative HBV DNA in metastasis and recurrence of hepatocellular carcinoma. J Med Virol.,2008,80(4):591-7.
42. TsuhoLa A, Arasc Y, Ren F, et al. Genotype may correlate with liver carcinogenesis and tumor characteristics in cirrhotic patients infected with hepatitis B Virus subtype adw.J Med Virol,2001,65:257-265.
43. Komita H, Homma S, Saotome H, et al. Interferon-γ produced by interleukin 12 activated tumor infiltrating CD8+T cells directly induces apoptosis of mouse hepatocellular carcinoma. J Hepatol,2006,45:662-7.
44. Taniguchi M, Kim SR, Imoto S, et al. Long-term outcome of percutaneous ethanol injection therapy for minimum-sized hepatocellular carcinoma.World J Gastroenterol,2008,14:1997-2002.
45.李波,陈汉.超声引导肝脏穿刺瘤内注射无水酒精治疗肝癌.中国实用外科杂志,1996,16(2)：84-85.
46.孙铁.超声导向注射无水酒精治疗肝癌50例报告.实用癌症杂志,1997,12(4)：307-308.
47.董晖,王朝杰.结直肠癌患者免疫状况与肿瘤分期的关系.国外医学：放射医学核医学分册,2005,29(3)：115-117.
48.皋岚湘,丁华野,邓永江,等.乳腺癌组织和Bcl-2、P53和c-erbB2蛋白表达和相互关系及意义.中华病理学杂志,1999,25(3)：165-168.
49. Momiyama K, Nagai H, Sumino Y. Changes of host immunity in relation to efficacy in liver cirrhosis patients with advanced hepatocellular carcinoma treated by intra-arterial chemotherapy[J]. Cancer Chemother Pharmacol,2009, 64(2):271-277.
50.董永华,林贵,王建华,等.介入治疗对原发性肝癌患者外周血T细胞亚群的影响[J].临床放射学杂志,1992,11：38.
51. Lakshmana Ayaru, Stephen P., Pereira. Unmasking of a-Fetoprotein-Specific
CD4+T Cell Responses in Hepatocellular Carcinoma Patients Undergoing Embolization.The Journal of Immunology,2007,178:1914-1922.
52. Martijn H, M. G. M, den Brok et al. In Situ Tumor Ablation Creates an Antigen Source for the Generation of Antitumor Immunity. Cancer Research,2004, 64:4024-4029.
53. Franco F, Bru C, Vilana R, et al. Percutaneous ethanol injection before liver transplantation in the hepatocellular carcinoma. Ann Hepatol,2009,8(3):220-7.
54. Lencioni RA, Allgaier HP, Cioni D, et al. Small hepatocellular carcinoma in cirrhosis:randomized comparison of radio-frequency thermal ablation versus percutaneous ethanol injection. Radiology,2003,228:235-240.
55. Schoppmeyer K, Weis S, Mossner J. Percutaneous ethanol injection or percutaneous acetic acid injection for early hepatocellular carcinoma. Cochrane Database Syst Rev,2009,3:6745-9.
56. Soo Ryang Kim, Susumu Imoto, Taisuke Nakajima. Well-differentiated hepatocellular carcinoma smaller than 15 mm in diameter totally eradicated with percutaneous ethanol injection instead of radiofrequency ablation. Hepatol Int, 2009,2:411-415.
57. Yoon I, Yim HJ, Kim JN, et al. A case of advanced hepatocellular carcinoma with portal vein tumor invasion controlled by percutaneous ethanol injection therapy. Korean Hepatol,2009,15:90-5.
58. ZHANG Liang, FAN Wei-jun, HUANG Jin-hua. Comprehensive sequential interventional therapy for hepatocellular carcinoma.Chinese. Medical Journal, 2009,19:2292-2298.
59. Taniguchi M, Kim S R, Imoto S, et al. Long-term outcome of percutaneous ethanol injection therapy for minimum-sized hepatocellular carcinoma[J]. World J Gastroenterol,2008,14(13):1997-2020.
60. Yoshida T, Okazaki N, Yoshino M, et al. Phase Ⅱ trial of high dose recombinant gamma-interferon in advanced hepatocellular carcinoma. Eur J Cancer,1990, 26(4):545-6.
61. Yang X, Zhang Y, Zhang L, et al. Silencing alpha-fetoprotein expression induces growth arrest and apoptosis in human hepatocellular cancer cell. Cancer Lett, 2008,271:281-93.
62. Li MS, Li PF, He SP, et al. Promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 line cells. World J Gastroenterol, 2002,8(3):469-471.
63. Li MS, Ma QL, Chen Q, et al. Alpha-fetoprotein triggers hepatoma cells escaping from immune surveillance through altering the expression of Fas/FasL and tumor necrosis factor related apoptosis inducing ligand and its receptor of lymphocytes and liver cancer cells.World J Gastroenterol,2005,11 (17):2564-25691.
64. Dudich E, Semenkova L, Dudich I,et al. Alpha-fetopmtein causes apoptosis in tumor cells via a pathway independent of CD95、TNF-R1 and TNF-R2 through activation of caspase-3 like proteases. Eur J Biochem,1999,66(3):750-761.
65. Butterfield LH, Koh A, Meng W,et al. Generation of human T cell responses to an HLA-A2.1-restricted peptide epitope from alpha-fetoprotein. Cancer Res, 1999,59:3134-3142.
66. Dranoff G, Mulligan RC. Gene transfer as cancer therapy. Adv Immunol,1995, 58:417-24..
67. Qiu G, Fang J, He Y.5'-CpG island methylation analysis identifies the MAGE-A1 and MAGE-A3genes as potential markers of HCC. Clinical Bio-chemistry,2006,39:259-266.
68. Zhang S, Lin ZN, Yang CF, Shi X, et al. Suppressed NF-κB and sustained JNK activation contribute to the sensitization effect of parthenolide to TNF-alpha-induced apoptosis in human cancer cells. Carcinogenesis.2004,25:2191-2199.
69. Liu J, Lin A. Role of JNK activation in apoptosis:a double-edged sword. Cell Res.,2005,15(1):36-42.
70. Chen F, Beezhold K, Castranova V. JNK1, a potential therapeutic target for hepatocellular carcinoma. Biochimica et Biophysica Acta,2009,1796:242-251.
2. Murgita A., Goidl S. α-Fetoprotein induces suppressor T cells in vitro. Nature, 1977,267:257.Gabrilovich D.Mechanisms and functional significance of tumour-induced dendritic-cell defects[J].Nat Rev Immunol,2004,4(12):941-952.
3. Peck A, Murgita R A., Wigzell H. Cellular and genetic restrictions in the immunoregulatory activity of α-fetoprotein. J Exp Med,1978,147:667-672.
4. Soon HU, Mulhall C, Alisa A, et al. Alpha-fetoprotein impairs APC function and induces their apoptosis.J Immunol,2004,173 (3):1772-1778.
5. Peng JR, Chen HS, Mou DC. Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters. Cancer Lett,.2005,219(2):223-32.
6. Caballero OL, Chen YT. Cancer/testis (CT) antigens:potential targets for immunotherapy.2009,100(11):2014-21.
7. Chen G, Luo D Nov-Dec; Expression of decoy receptor 3 in liver tissue microarrays. Natl Med J India,2008,21(6):275-8.
8. Chen C, Zhang C, Zhuang G..Decoy receptor 3 overexpression and immunologic tolerance in hepatocellular carcinoma (HCC) developme.Cancer Invest,2008 D, 26(10):965-74.
9. Yang C, Hsieh S L, Teng C M, et al. Soluble Decoy Receptor 3 Induces Angiogenesis by Neutralization of TL1A, a Cytokine Belonging to Tumor Necrosis Factor Superfamily and Exhibiting Angiostatic Action.Cancer Res, 2004,64:1122-1129.
10. Yang C R, Hsieh S L, Ho F M. Decoy Receptor 3 increases monocyte adhesion to endothelial cells via NF-κB dependent up-regulation of Intercellular Adhesion Molecule-1, VCAM-1, and IL-8 expression. The Journal of Immunology,2005, 174:1647-1656.
11. Yang XR, Xu Y, Yu B, et al. High expression levels of putative hepatic stem/ progenitor cell biomarkers related to tumour angiogenesis and poor prognosis of
hepatocellular carcinoma. Gut.,2010 May 4. [Epub ahead of print]
12. Sutton A, Friand V, Brule-Donneger S, et al.Stromal cell-derived factor-1/ chemokine (C-X-C motif) ligand 12 stimulates human hepatoma cell growth, migration, and invasion. Mol Cancer Res,2007,5:21-33.
13. Liu H,Pan Z,Li A,et al. Roles of chemokine receptor 4 (CXCR4) and chemokine ligand 12 (CXCL12) in metastasis of hepatocellular carcinoma cells. Cell Mol Immunol.2008,5:373-378.
14. Chu H, Zhou H, Liu Y, et al. Functional expression of CXC chemokine recepter-4 mediates the secretion of matrix metalloproteinases from mouse hepato-carcinoma cell lines with different lymphatic metastasis ability. Int J Biochem Cell Biol,2007,39:197-205.
15. Rubie C, Frick VO, Wagner M, et al.Chemokine expression in hepatocellular carcinoma versus colorectal liver metastases. World J Gastroenterol,2006,12: 6627-6633.
16. Rubie C, Frick VO, Wagner M, et al. Enhanced expression and clinical significance of CC-chemokine MIP-3 alpha in hepatocellular carcinoma.Scand J Immunol,2006,63:468-477.
17. Matsubara T, Ono T, Yamanoi A, et al. Fractalkine-CX3CR1 axis regulates tumor cell cycle and deteriorates prognosis after radical resection for hepatocellular carcinoma. J Surg Oncol,2007,95:241-249.
18. Miihlbauer M, Ringel S, Hartmann A, et al.Lack of association between the functional CX3CR1 polymorphism V249I and hepatocellular carcinoma. Oncol Rep,2005,13:957-963.
19. Nagaoka S, Yoshida T, Akiyoshi J, et al. The ratio of serum placenta growth factor to soluble vascular endothelial growth factor receptor-1 predicts the prognosis of hepatocellular carcinoma. Oncol Rep,2010,23(6):1647-54.
20. Heneghan MA. Frequency and nature of cytokine gene polymorphisms in hepato-cellular carcinoma in Hong Kong Chinese. Int J Gastrointest.Cancer,2003,34: 19-26.
21. Shin HD. Interleukin 10 haplotype associated with increased risk of hepatocellular carcinoma. Hum Mol Genet,2003,12,901-906.
22. Simona Ognjanovic, Jian-Min Yuan, Ann K.Genetic polymorphisms in the cytokine genes and risk of hepatocellular carcinoma in low-risk non-Asians of USA. Carcinogenesis,2009,30(5):758-762.
23. Dong Z Z, Yao D F, Yao M, et al.Clinical impact of plasmaTGF betal and circulatingTGF betal mRNA in diagnosis of hepatocellular carcinoma. Hepatobiliary [J].Pancreat Dis Int,2008,7(3):288 295.
24. Benetti A, Berenzi A, Gambarotti M, et al.Transforming growth factor-betal and CD105 promote the migration of hepatocellular carcinoma-derived endothelium. Cancer Res,2008,15,68(20):8626-34.
25. Chen J, Zheng XH, Tang XP, et al.A comparative study of serum sFas in patients with hepatocellular cancer and chronic hepatitis]. Hunan Yi Ke Da Xue Xue Bao,2001,26:173-174.
26. Rahman N, Hanaa M. Serum levels of soluble Fas, soluble tumor necrosis factor-receptor Ⅱ, interleukin-2 receptor and interleukin-8 as early predictors of hepatocellular carcinoma in Egyptian patients with hepatitis C virus genotype-4. Comp Hepatol,2010,9:1-5.
27. Shi F,Shi M,Zeng Z,et al. PD-1 and PD-L1 upregulation promotes CD8(+)T-cell apoptosis and post-operative recurrence in hepatocellular carcinoma patients.Int J Cancer,2010,4:19-24.
28. NakamotoY, Kaneko S. Dendritic cell-based immunotherapy for hepatocellular carcinoma. Gan To Kagaku Ryoho,2010,37(3):413-6.
29. Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance[J]. Nat Rev Cancer,2005,5(4):263-274.
30. Shevach EM, DiPaolo RA, Andersson J, et al.The lifestyle of naturally occurring CD4+CD25+FoxP3+regulatory T cells[J].Immunol Rev,2006,212:60-73.
31. Shevach EM, DiPaolo RA, Andersson J, et al.The lifestyle of naturally occurring CD4+CD25+FoxP3+regulatory T cells [J].Immunol Rev,2006,212:60-73.
32. Enarsson K, Johnsson E, Lindholm C, et al. Differential mechanisms for T lymphocyte recruitmentin normal and neoplastic human gastric mucosa.[J] Clin Immunol,2006,118(1):24-34.
33. Enarsson K, Johnsson E, Lindholm C, et al. Differential mechanisms for T lymphocyte recruitmentin normal and neoplastic human gastric mucosa.[J] Clin Immunol,2006,118(1):24-34.
34. Kobayashi N, Hiraoka N, Yamagaini W, et al.FOXP3+Regulatory T cells affect the development and progression of hepatocarcinogenesis. Clin Cancer Res, 2007,13(3):902-911.
35. Zeng Z, Yao W, Xu X, et al. Hepatocellular carcinoma cells deteriorate the biophysical properties of dendritic cells. Cell Biochem Biophys,2009, 55(1):33-43.
36. Chew V, Tow C, Teo M, et al. Inflammatory tumour microenvironment is associated with superior survival in hepatocellular carcinoma patients.J Hepatol, 2010,52(3):370-9.
37. Nagao M, Nakajima Y, Hisanaga M, et al. The alteration of Fas receptor and ligand system in hepatocellular carcinomas:how do hepatoma cells escape from the host immune surveillance in vivo? Hepatology,1999,30(2):413-21.
38. Dranoff G, Mulligan RC. Gene transfer as cancer therapy. Adv Immunol.1995, 58:417-54.
39. Beatty GL, Paterson Y. IFN-gamma can promote tumor evasion of the immune system in vivo by down-regulating cellular levels of an endogenous tumor antigen[J]. Immunol,2000,165:5502-5508
40. Le Poole IC, Riker AI, Quevedo ME, et al.Interferon-gammar reduces melanosomal antigen expression and recognition of melanoma cells by cytotoxic T. [J]. Am J Pathol,2002,160:521-528
41. He F, Wang XH, Zhang GM, et al. Sustained low-level expression of interferon-γ promotes tumor development:Potential insights in tumor prevention and tumor immunotherapy. Cancer Immunol Immunother,2005,54:891-897.
1. Sean F, Altekruse, Katherine A, el al. Hepatocellular Carcinoma Incidence, Mortality, and Survival Trends in the United States From 1975 to 2005. J Clin Oncol,2009,27(9):1485-1491.
2. Rampone B, Schiavone B, Martino A, el al. Current management strategy of hepatocellular carcinoma.World J Gastroenterol,2009,15(26):3210-3216.
3. Yau T, Chan P, Epstein R, el al. Evolution of systemic therapy of advanced hepatocellular carcinoma.World J Gastroenterol.2008,14(42):6437-6441.
4. Choi D, Lim H, Lee WJ, el al. Radiofrequency Ablation of Liver Cancer:Early Evaluation of Therapeutic Response with Contrast-Enhanced Ultrasonography. Korean J Radiol.2004,5(3):185-198.
5. Lin SM, Lin CJ, Lin CC, el al. Randomised controlled trial comparing percutaneous radiofrequency thermal ablation, percutaneous ethanol injection, and percutaneous acetic acid injection to treat hepatocellular carcinoma of 3 cm or less. Gut,2005,54(8):1151-1156.
6. Llovet JM, Real MI, Montana X, el al. Arterial embolisation or chemo-embolisation versus symptomatic treatment in patients with unresectable hepatocellular carcinoma:a randomised controlled trial. Lancet,2002,59: 1734-1739.
7. Zhang W, Liu J, Wu Y, el al. Immunotherapy of hepatocellular carcinoma with a vaccine based on xenogeneic homologous a fetoprotein in mice. Biochemical and Biophysical Research Communications.2008,376:10-14.
8. Sarobe P, Feijoo'E, Alfaro C, el al. MAGE antigens:therapeutic targets in hepatocellular carcinoma?. Journal of Hepatology,2004,40(1):155-158.
9. Greten TF, Manns MP, Korangy F. Immunotherapy of hepatocellular carcinoma. Journal of Hepatology,2006,45:868-878.
10. Porta C, Larghi P, Rimoldi M, el al.Cellular and molecular pathways linking inflammation and cancer. Immunobiology,2009,214:761-77.
11. McGlynn KA, London WT. Epidemiology and natural history of hepatocellular carcinoma. Best Pract Res Clin Gastroenterol,2005,19:3-23.
12. Chisari FV, Isogawa M, Wieland SF. Pathogenesis of hepatitis B virus infection. Pathol Biol (Paris),2010. [Epub ahead of print]
13. Nardin A. Abastado JPFront Biosci. Macrophages and cancer,2008,13:3494-505.
14. Okada T, Sada T, Kubota K. Deferoxamine enhances anti-proliferative effect of interferon-γ against hepatocellular carcinoma cells.Cancer Lett,2007,248: 24-31.
15. Kremsdorf D, Soussan P, Paterlini-Brechot P, el al.Hepatitis B virus related hepatocellular carcinoma:paradigms for viral-related human carcinogenesis. Oncogene,2006,25:3823-3833.
16. Tu Z, Bozorgzadeh A, Pierce RH, el al. TLR-dependent cross talk between human Kupffer cells and NK cells. J Exp Med,2008,205:233-244.
17. Chen Y M, Yang W K, Whany P J, et al. Restoration of the immunocompetence by IL-2 activation and TCR-CD3 engagement of the in vivo anergized tumor-specific CTL from lung cancer patients [J]. Immunother,1997,20:354-358.
18. Sal B, Marotta A, Matthewson C, el al. Investigation of the Mek-MAPkinase-Rsk pathway in human breast cancer. Anticancer Res,1999,19:731-740.
19. Shimizu Y, Iwatsuki S, Herberman R et al. Effects of cytokines on in vitro growth of tumor-infiltrating lymphocytes obtained from human primary and metastatic liver tumors. Cancer Immunol. Immunother,1991,32:280-288.
20. Nakazaki H. Preoperative and postoperative cytokines in patients with cancer[J]. Cancer,1992,70:709.
21. Hus U, Lees B, Kinc M, et al. Papid recruitment of macrophages in interleukin-12-mediated tumor regression. Immunology,1998,95:156-163.
22. Lars A Ormandy, Anatol Farber, Tobias Cantz et al. Direct ex vivo analysis of dendritic cells in patients with hepatocellular carcinoma. World J Gastroenterol. 2006,12(20):3275-3282.
23. Um SH, Mulhall C, Alisa A, et al. Alpha-fetoprotein impairs APC function and induces their apoptosis. J Immunol.2004,173:1772-1778.
24. Nishioka Y, Wen H, Mitani K et al. Differential effects of IL-12 on the generation of alloreactive CTL mediated by murine and human dendritic cells:a critical role for nitric oxide. LeucocBiol,2003,73:621-629.
25. Lo CH, Chang CM, Tang SW, et al. Differential antitumor effect of interleukin-12 family cytokines on orthotopic hepatocellular carcinoma. Gene Med,2010, 12(5):423-34.
26. Whiteside TL, Herberman RB. The role of natural killer cells in human disease. Clin Immunol Immunopathol,1989,53:1-23.
27. Maione S, Lamberti L. Nucleolar organizer regions activity in lymphocytes of patientswith laryngeal carcinoma. Soc Ital Biol Sper,1993,69:741.
28. Norder H, Courouce AM, Coursaget P, et al.Genetic diversity of hepatitis B virus strains derived worldwide:genotypes, subgenotypes, and HBsAg subtypes. Intervirology,2004,47:289-309.
29. Pujol FH, Navas MC, Hainaut P, et al. Worldwide genetic diversity of HBV genotypes and risk of hepatocellular carcinoma. Cancer Lett,2009,286(1):80-88.
30. Raimondi S, Maisonneuve P, Bruno S, et al.Is response to antiviral treatment influenced by hepatitis B virus genotype?. J Hepatol,2010,52:441-9.
31.Yu MW, Yeh SH, Chen PJ, et al. Hepatitis B virus genotype and DNA leveland hepatocellular carcinoma:a prospective study in men. J Natl Cancer Inst,2005, 97(4):265-72.
32. Kao JH, Chen PJ, Lai MY, et al.Genotypes and clinical phenotypesof hepatitis B virus in patients with chronic hepatitis B virus infection. J Clin Microbiol, 2002,40(4):1207-9.
33. Kao JH, Chen PJ, Lai MY, et al. Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers.J Med Virol,2004,72(3):63-9.
34. Pujol FH, Navas MC, Hainaut P, et al.Worldwide genetic diversity of HBV genotypes and risk of hepatocellular carcinoma. Cancer Lett,2009,286(1):80-8.
35. Ding X, Mzokami M, Yao G, et al. Hepatitis B virus genotype distribution among chronic hepatitis B carriers china shanghaj.[J] Interviology,2001,44(1):43-46.
36. Orito E, Ichida T, Sakugawa H, et al.Geographic distribution ofhepatitis B virus genotype in patients with chronic HBV infection in Japans. [J] Hepatology, 2001,34(3):590-594.
37. Yang HI, Yeh SH, Chen PJ, et al. Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma J. Natl Cancer Inst.2008,100(16):1134-1143.38、Kao JH, Chen PJ, Lai MY, et al. Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers. Gastroenterology,2003,124 (2):327-34.
38. Orito E, Mizokami M, Sakugawa H, et al. A case-control study for clinical and molecular biological differences between hepatitis B viruses of genotypes B and C. Hepatology,2001,33(1):218-23.
39. Ding X, Mizokami M, Ge X, et al.Different hepatitis B virus genotype distributions among asymptomatic carriers and patients with liver diseases [J] Hepatol Res,2002,22(1):37-44.
40. Gaglio P, Singh S, Degertekin B, et al. Impact of the hepatitis B virus genotype on pre-and post-liver transplantation outcomes. Liver Transpl.,2008,14(10): 1420-7.
41. Huang Y, Wang Z, An S, et al. Role of hepatitis B virus genotypes and quantitative HBV DNA in metastasis and recurrence of hepatocellular carcinoma. J Med Virol.,2008,80(4):591-7.
42. TsuhoLa A, Arasc Y, Ren F, et al. Genotype may correlate with liver carcinogenesis and tumor characteristics in cirrhotic patients infected with hepatitis B Virus subtype adw.J Med Virol,2001,65:257-265.
43. Komita H, Homma S, Saotome H, et al. Interferon-γ produced by interleukin 12 activated tumor infiltrating CD8+T cells directly induces apoptosis of mouse hepatocellular carcinoma. J Hepatol,2006,45:662-7.
44. Taniguchi M, Kim SR, Imoto S, et al. Long-term outcome of percutaneous ethanol injection therapy for minimum-sized hepatocellular carcinoma.World J Gastroenterol,2008,14:1997-2002.
45.李波,陈汉.超声引导肝脏穿刺瘤内注射无水酒精治疗肝癌.中国实用外科杂志,1996,16(2)：84-85.
46.孙铁.超声导向注射无水酒精治疗肝癌50例报告.实用癌症杂志,1997,12(4)：307-308.
47.董晖,王朝杰.结直肠癌患者免疫状况与肿瘤分期的关系.国外医学：放射医学核医学分册,2005,29(3)：115-117.
48.皋岚湘,丁华野,邓永江,等.乳腺癌组织和Bcl-2、P53和c-erbB2蛋白表达和相互关系及意义.中华病理学杂志,1999,25(3)：165-168.
49. Momiyama K, Nagai H, Sumino Y. Changes of host immunity in relation to efficacy in liver cirrhosis patients with advanced hepatocellular carcinoma treated by intra-arterial chemotherapy[J]. Cancer Chemother Pharmacol,2009, 64(2):271-277.
50.董永华,林贵,王建华,等.介入治疗对原发性肝癌患者外周血T细胞亚群的影响[J].临床放射学杂志,1992,11：38.
51. Lakshmana Ayaru, Stephen P., Pereira. Unmasking of a-Fetoprotein-Specific
CD4+T Cell Responses in Hepatocellular Carcinoma Patients Undergoing Embolization.The Journal of Immunology,2007,178:1914-1922.
52. Martijn H, M. G. M, den Brok et al. In Situ Tumor Ablation Creates an Antigen Source for the Generation of Antitumor Immunity. Cancer Research,2004, 64:4024-4029.
53. Franco F, Bru C, Vilana R, et al. Percutaneous ethanol injection before liver transplantation in the hepatocellular carcinoma. Ann Hepatol,2009,8(3):220-7.
54. Lencioni RA, Allgaier HP, Cioni D, et al. Small hepatocellular carcinoma in cirrhosis:randomized comparison of radio-frequency thermal ablation versus percutaneous ethanol injection. Radiology,2003,228:235-240.
55. Schoppmeyer K, Weis S, Mossner J. Percutaneous ethanol injection or percutaneous acetic acid injection for early hepatocellular carcinoma. Cochrane Database Syst Rev,2009,3:6745-9.
56. Soo Ryang Kim, Susumu Imoto, Taisuke Nakajima. Well-differentiated hepatocellular carcinoma smaller than 15 mm in diameter totally eradicated with percutaneous ethanol injection instead of radiofrequency ablation. Hepatol Int, 2009,2:411-415.
57. Yoon I, Yim HJ, Kim JN, et al. A case of advanced hepatocellular carcinoma with portal vein tumor invasion controlled by percutaneous ethanol injection therapy. Korean Hepatol,2009,15:90-5.
58. ZHANG Liang, FAN Wei-jun, HUANG Jin-hua. Comprehensive sequential interventional therapy for hepatocellular carcinoma.Chinese. Medical Journal, 2009,19:2292-2298.
59. Taniguchi M, Kim S R, Imoto S, et al. Long-term outcome of percutaneous ethanol injection therapy for minimum-sized hepatocellular carcinoma[J]. World J Gastroenterol,2008,14(13):1997-2020.
60. Yoshida T, Okazaki N, Yoshino M, et al. Phase Ⅱ trial of high dose recombinant gamma-interferon in advanced hepatocellular carcinoma. Eur J Cancer,1990, 26(4):545-6.
61. Yang X, Zhang Y, Zhang L, et al. Silencing alpha-fetoprotein expression induces growth arrest and apoptosis in human hepatocellular cancer cell. Cancer Lett, 2008,271:281-93.
62. Li MS, Li PF, He SP, et al. Promoting molecular mechanism of alpha-fetoprotein on the growth of human hepatoma Bel7402 line cells. World J Gastroenterol, 2002,8(3):469-471.
63. Li MS, Ma QL, Chen Q, et al. Alpha-fetoprotein triggers hepatoma cells escaping from immune surveillance through altering the expression of Fas/FasL and tumor necrosis factor related apoptosis inducing ligand and its receptor of lymphocytes and liver cancer cells.World J Gastroenterol,2005,11 (17):2564-25691.
64. Dudich E, Semenkova L, Dudich I,et al. Alpha-fetopmtein causes apoptosis in tumor cells via a pathway independent of CD95、TNF-R1 and TNF-R2 through activation of caspase-3 like proteases. Eur J Biochem,1999,66(3):750-761.
65. Butterfield LH, Koh A, Meng W,et al. Generation of human T cell responses to an HLA-A2.1-restricted peptide epitope from alpha-fetoprotein. Cancer Res, 1999,59:3134-3142.
66. Dranoff G, Mulligan RC. Gene transfer as cancer therapy. Adv Immunol,1995, 58:417-24..
67. Qiu G, Fang J, He Y.5'-CpG island methylation analysis identifies the MAGE-A1 and MAGE-A3genes as potential markers of HCC. Clinical Bio-chemistry,2006,39:259-266.
68. Zhang S, Lin ZN, Yang CF, Shi X, et al. Suppressed NF-κB and sustained JNK activation contribute to the sensitization effect of parthenolide to TNF-alpha-induced apoptosis in human cancer cells. Carcinogenesis.2004,25:2191-2199.
69. Liu J, Lin A. Role of JNK activation in apoptosis:a double-edged sword. Cell Res.,2005,15(1):36-42.
70. Chen F, Beezhold K, Castranova V. JNK1, a potential therapeutic target for hepatocellular carcinoma. Biochimica et Biophysica Acta,2009,1796:242-251.