树突状细胞与肝细胞癌免疫治疗

树突状细胞（DC）是体内功能最强的抗原提呈细胞（APC）,由DC激活的T细胞免疫在抗肿瘤过程中起着主导作用。关于DC肿瘤疫苗的基础与临床研究结果显示,DC疫苗在恶性肿瘤治疗中具有良好的应用前景。     

树突状细胞与肝细胞癌免疫治疗

树突状细胞(DC)是体内功能最强的抗原提呈细胞(APC),由DC激活的T细胞免疫在抗肿瘤过程中起着主导作用。关于DC肿瘤疫苗的基础与临床研究结果显示,DC疫苗在恶性肿瘤治疗中具有良好的应用前景。     

树突状细胞与肝癌的免疫治疗

 近二十年来通过对树突状细胞（dendritic cell，DC）的研究，学者们一致认为DC是目前发现的功能最强的抗原提呈细胞（antigenpresentingcell，APC），DC是启动、调控、并维持免疫应答的中心环节。肝癌的免疫治疗一直是研究的热点，以DC为基础的肝癌的免疫治疗日益受到重视，取得了初步的成果。本文就DC在肿瘤免疫中的作用机制及应用于HCC（hepatocellular carcinoma，HCC）免疫治疗的可能性、研究现状和成果作一综述。     

树突状细胞免疫治疗转移性肾细胞癌临床转化的现状

树突状细胞（dendritic cell,DC）免疫治疗作为一种无严重毒性反应、前景广阔的主动免疫治疗策略,自从1998年以来,已经在转移性肾细胞癌（metastatic renal cell carcinoma,mRCC）的治疗中进行了广泛研究,并在2007年5月韩国FDA批准了一种称为CreaVax RCC的自体DC疫苗用于mRCC的治疗。目前已经有31项非随机对照的Ⅰ/Ⅱ期临床试验研究了DC免疫治疗用于mRCC患者的安全性和有效性,虽然这些试验结果已经证实DC免疫治疗的安全性和对一小部分mRCC患者的有效性,但这些临床试验在受试者选择、DC疫苗的标准化、免疫学终点和临床终点的选择和评价等方面难以统一标准,这导致DC免疫治疗疗效的发挥受限且不同试验结果无法进行直接比较;另外,所开展的试验均为非随机对照小样本临床研究。因此,期待临床试验设计方案的进一步优化,以客观评估DC免疫治疗对mRCC患者的临床治疗效果,并应在临床试验中加强对DC免疫治疗联合mRCC一线治疗方案疗效的评价,以进一步开发对mRCC更加有效的联合治疗策略。     

肝细胞癌和肝内胆管细胞癌流行病学研究进展

摘 要：原发性肝癌在全球恶性肿瘤死因中位居第4位。肝细胞癌和肝内胆管细胞癌是其最常见的两种病理类型。虽两者均属原发性肝癌,但在病因学、流行病学、临床诊疗与预后等方面均有差别。该文就近年来肝细胞癌和肝内胆管细胞癌的流行病学研究进展作简要综述。     

肝脏原发性鳞状细胞癌1例

1　病案摘要　　患者女 ,4 0岁 ,已婚。畏寒、发热、纳差伴左季肋部隐痛1月余 ,体温 38 5℃ ,外院Ｂ超检查发现肝脏脓肿 ,皮肤无黄染 ,经抗炎治疗无效于 1992年 8月来我院住院治疗。专科检查 :心、肺、脾、胃镜、肠镜检查均无异常。腹平软 ,肝肋下7ｃｍ ,肝区有压痛、叩击痛。Ｂ超检查示肝内占位 ,肝脓肿可能。皮肤、眼结膜无黄染。血ＡＦＰ阴性 ,肝功无异常。行剖腹探查术 ,术中见肝脏内有一肿瘤 ,直径约 7ｃｍ ,遂将病变肝叶切除。2　病理检查　　巨检 :肝叶切除标本 ,大小 2 0ｃｍ× 15ｃｍ× 10 5ｃｍ ,切面见肝叶内有一肿瘤 ,大小 7 3…     

肝星状细胞和肝细胞癌的相互作用

肝星状细胞(HSC)是肝纤维化及正常肝脏中细胞外基质的主要来源.慢性肝损伤时,HSC发生表犁变化转变成肌纤维母细胞,引起肝纤维化.肝细胞癌是多因素疾病,基质中有明显星状细胞浸润.探讨肝细胞癌与HSC相互作用关系可为肝细胞癌的治疗提供新思路.     

大肠癌树突状细胞和癌周淋巴细胞反应的关系

用Ｓ－１００蛋白质抗体ＡＢＣ免疫组化法定量检测１４４例大肠癌组织树突状细胞（ＤＣ），同时观察癌周淋巴细胞反应。结果表明ＤＣ在癌组织中和淋巴细胞相伴浸润，癌周淋巴细胞反应明显者，癌组织ＤＣ数量明显增多，肿瘤浸润至浆膜层及淋巴结转移者ＤＣ明显减少。提示ＤＣ在辅助Ｔ淋巴细胞免疫，增强机体抗肿瘤免疫力方面有重要作用。     

腹腔镜与开腹肝脏切除术治疗肝细胞癌的临床疗效分析

  目的  探讨腹腔镜与开腹肝脏切除术治疗肝细胞癌（hepatocellular carcinoma,HCC）的近期及远期临床疗效。  方法  回顾性分析2013年1月至2016年5月在福建医科大学附属第一医院行肝细胞癌切除患者的临床资料,为减少选择偏倚采用逐一配对法进行配对,最终纳入腹腔镜组105例,开腹组105例。比较手术及术后的无瘤生存率以及总生存率,并行统计学分析。  结果  腹腔镜组术后住院时间更短[（8.68±2.82）d vs.（10.61±2.95）d,P < 0.01],肝门阻断率更低（20.0% vs. 41.0%,P < 0.01）,且腹腔引流管拔除时间更早[（4.45±2.53）d vs.（5.40±2.43）d,P < 0.01]。腹腔镜组1、2、3年生存率分别为96.88%、87.54%、79.50%,开腹组1、2、3年生存率分别为94.91%、86.29%、76.37%（P=0.670）。腹腔镜组1、2、3年无瘤生存率分别为72.09%、60.16%、52.08%,开腹组1、2、3年无瘤生存率分别为69.48%、56.50%、48.13%（P=0.388）。  结论  腹腔镜肝脏切除术（laparoscopic liver resection,LLR）治疗肝细胞癌安全可行,与开腹肝脏切除术（open liver resection,OLR）相比,具有相当的远期疗效,同时腔镜组术后住院时间更短、肝门阻断率更低,且引流管拔除时间更早,显示较好的近期疗效。      

NY-ESO-1致敏树突状细胞诱导的CTL对肝癌细胞株的特异性杀伤作用

目的:探讨肿瘤睾丸抗原NY-ESO-1(New York-esophageal-1)致敏树突状细胞体外诱导特异性CTL对肝癌细胞株的杀伤作用.方法:重组质粒pGEX-ESO1经原核诱导表达并纯化GST-ESO1融合蛋白肽.重组人粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)和白细胞介素4(rhIL-4)诱导培养人外周血来源的树突状细胞(dendritic cells, DCs),经GST-ESO1融合蛋白肽致敏后诱导特异性CTL增殖.以此CTL为效应细胞,分别以NY-ESO-1阳性表达的肝癌细胞株HepG2和不表达NY-ESO-1的肝癌细胞株H2P为靶细胞,MTT法检测CTL对肝癌细胞株的杀伤作用.结果:重组质粒pGEX-ESO1经IPTG诱导,在大肠杆菌中表达相对分子质量约36 000的GST-ESO1融合蛋白肽,纯化后的质量浓度为50 μg/ml;经rhGM-CSF和rhIL-4联合诱导成功培养人外周血DCs,其表型分子HLA-DR为91.4%、CD86为70.5%、CD83为71.2%、CD80为55.3%.NY-ESO-1致敏的DCs能明显诱导CTL增殖,此CTL对肝癌细胞株HepG2的杀伤率显著高于GST刺激组、未致敏DC组和无DC刺激组(均P<0.05),效靶比为50 ∶1时杀伤效应达到最高峰[(53.23±3.78)%,P<0.01];相同条件下CTL对H2P细胞无特异性杀伤作用.结论: NY-ESO-1抗原致敏的DCs在体外可诱导同种CTL产生和增殖,后者对NY-ESO-1阳性肝癌细胞株具有特异性杀伤效应,该方法为肝癌免疫治疗提供了一条新思路.     

Hepatic iron overload and hepatocellular carcinoma

The liver is the main storage site for iron in the body. Excess accumulation of iron in the liver has been well-documented in two human diseases, hereditary hemochromatosis and dietary iron overload in the African. Hepatic iron overload in these conditions often results in fibrosis and cirrhosis and may be complicated by the development of hepatocellular carcinoma. Malignant transformation usually occurs in the presence of cirrhosis, suggesting that free iron-induced chronic necroinflammatory hepatic disease plays a role in the hepatocarcinogenesis. However, the supervention of hepatocellular carcinoma in the absence of cirrhosis raises the possibility that ionic iron may also be directly hepatocarcinogenic. Support for this possibility is provided by a recently described animal model of dietary iron overload in which iron-free preneoplastic nodules and hepatocellular carcinoma developed in the absence of fibrosis or cirrhosis. The mechanisms by which iron induces malignant transformation have yet to be fully characterized but the most important appears to be the generation of oxidative stress. Free iron generates reactive oxygen intermediates that disrupt the redox balance of the cells and cause chronic oxidative stress. Oxidative stress leads to lipid peroxidation of unsaturated fatty acids in membranes of cells and organelles. Cytotoxic by-products of lipid peroxidation, such as malondialdehyde and 4-hydroxy-2′-nonenal, are produced and these impair cellular function and protein synthesis and damage DNA. Deoxyguanosine residues in DNA are also hydroxylated by reactive oxygen intermediates to form 8-hydroxy-2′-deoxyguanosine, a major promutagenic adduct that causes G:C to T:A transversions and DNA unwinding and strand breaks. Free iron also induces immunologic abnormalities that may decrease immune surveillance for malignant transformation.     

Administration of dendritic cells in cancer nodules in hepatocellular carcinoma

Dendritic cells (DCs), the most potent antigen-presenting cells in vivo, are now used for cancer immunotherapy during which they are usually administered to the blood of patients with cancer. However, the route of administration of DCs affects the magnitude of immune responses. This study was conducted to assess the safety of the direct administration of DCs into cancer nodules. DCs were generated by culturing peripheral blood mononuclear cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 7 days. After confirming the phenotype and function, one hundred thousand DCs were injected directly into the cancer nodules of 4 patients with hepatocellular carcinoma (HCC) under ultrasonography guidance 48 h after the administration of 100% ethanol. All patients were monitored for any alteration in generalized condition, signs of inflammation, and liver and kidney function for the next 14 days. In addition, the final assessment of the safety of the administration of DCs into cancer nodules was performed 6 months after therapy commencement. The injection of 100% ethanol disrupted the HCC nodules in all 4 patients. DCs were distributed uniformly in the cancer nodules as assessed by ultrasonography. The administration of DCs into cancer nodules was well tolerated by all patients and there were no immediate or delayed side effects. The tumor marker decreased in one patient after the direct administration of DCs. Direct administration of DCs into the cancer nodules of patients with HCC was safe.     

以AFP为靶点的肝癌树突状细胞免疫治疗的实验研究

目的探讨AFP作为肝癌基因治疗和免疫治疗靶点的可行性以及AFP基因转染的树突状细胞（AFP-DC）疫苗对表达AFP肝细胞癌的免疫治疗作用.方法构建AFP-cDNA 真核表达载体,体外转染DC,制备AFP-DC瘤苗,诱导针对表达AFP的肝癌细胞株HepG2的特异性免疫反应,MTT法检测效应细胞对靶细胞的杀伤率.结果 AFP-DC瘤苗能够分泌AFP抗原,培养上清中AFP含量为0.8805 IU/ml,与空载体组和空白对照组相比,差异显著（P〈0.05）.免疫荧光检测可见AFP-DC胞浆及胞膜有AFP抗原分子表达;活化的CTL能够对表达AFP肝癌细胞起特异性杀伤作用,杀伤效率可达84.05%,与空载体组和空白对照组比较,差异显著（P〈0.05）.结论 AFP作为靶点治疗肝癌具有可行性,AFP可作为肝癌靶向治疗的新的突破点;AFP-DC疫苗可以作为表达AFP肝癌的一种免疫治疗手段,为DC瘤苗的临床应用奠定了基础.     

Immune responses of dendritic cells combined with tumor-derived autophagosome vaccine on hepatocellular carcinoma

Background

To induce and collect tumor-derived autophagosomes (DRibbles) from tumor cells as an antitumor vaccine by inhibiting the functions of proteasomes and lysosomes.

Methods

Dendritic cells (DCs) generated from peripheral blood mononuclear cell (PBMC) of hepatocellular carcinoma (HCC) patients were cocultured with DRibbles, and then surface molecules of DCs, as well as surface molecules on DCs, were determined by flow cytometry. Meanwhile, immune responses of the DCs-DRibbles were examined by mixed lymphocyte reactions.

Results

DRibbles significantly induced the expression of CD80, CD83, CD86 and HLA-DR on DCs. The enzyme-linked immunosorbnent assay (ELISA) showed that IFN-γ levels after vaccination increased than before in most patients, but CD8+ proportion of PBMC increased only in nine patients. Higher levels of IFN-γ were detected in the CD8+ cells than CD4+ T cells. These results suggested that DCs-DRibbles vaccine could induce antigen-specific cellular immune response on HCC and could prime strong CD8+ T cell responses, supporting it as a tumor vaccine candidate.

Conclusions

Our results demonstrate that HCC/DRibbles-pulsed DCs immunotherapy might be deployed as an effective antitumor vaccine for HCC immunotherapy in clinical trials.     

Hepatic resection for hepatocellular carcinoma in Taiwan.

AIMS: We examined the clinical features and outcome of 80 patients with hepatocellular carcinoma (HCC) who had undergone hepatic resection; 32 patients were under 30 years old (younger-HCC), and 48 patients were over 70 years (elderly-HCC).METHODS: Clinical features of 32 cases of younger-HCC (less than 30 years old) and 48 elderly-HCC (over 70 years old) were reviewed between 1986 and 1999. The clinical features, pathological findings and outcome of the younger and elderly HCC patients were summarized for comparison.RESULTS: Of 573 HCC patients who underwent hepatic resection, 32 were younger than 30 years old and 48 older than 70 years. Less hepatitis B infection, higher hepatitis C infection, more liver cirrhosis development, and a higher ICG 15min retention ratio than the younger group. The younger group tended to have a larger tumor size, more major hepatectomy, more operative blood loss during operation, more perioperative blood transfusion, and more recurrence when compared with the elderly-HCC group. The elderly-HCC group had a significantly better disease-free survival rate than the younger-HCC group. The younger-HCC group had a similar overall survival rate to the elderly-HCC group.CONCLUSIONS: These results seem to indicate a possible difference in the HCC carcinogenesis between younger and elderly patients. The overall survival of the younger HCC patients who undergone hepatic resection was as favorable as that of the elderly.     

Hepatic resection for hepatocellular carcinoma exceeding Milan criteria

BackgroundMany hepatocellular carcinomas (HCCs) are discovered at an advanced stage. The efficacy of transplantation for such tumors is doubtable. The aim of this retrospective study was to determine liver resection efficacy in patients with large HCC regarding long term and disease- free survival.MethodsBetween 2002 and 2008, sixty six patients with large HCC (>5 cm) underwent hepatectomy. Fifty nine patients had background cirrhosis due to hepatitis B, C or other reason and preserved liver function (Child A). Liver function was assessed by both Child's–Pugh grading and MELD score. Conventional approach of liver resection was performed in most cases.ResultsThe 5-year overall survival was 32% with a median follow up of 33 months. The three year disease-free survival was 33% in our cohort. On multivariate analysis, only tumor size and grade remained independent predictors of adverse long term outcome. Multivariate analysis identified size of the primary tumour and degree of differentiation as risk factors for recurrence. Median blood loss was 540 ml and median transfusion requirements were two units of pack red blood cells. Morbidity included pleural effusion (n = 18), biliary fistula (n = 4), peri-hepatic abscess (n = 4), hyperbilirubinemia (n = 3), pneumonia (n = 5) and wound infection (n = 6). No peri-operative mortality was reported in our study.ConclusionPartial hepatectomy is safe in selective patients with large HCC. Surgical resection if feasible is suggested in patients with large HCC because it prolongs both overall and disease-free survival with low morbidity.     

Lentivirally engineered dendritic cells activate AFP-specific T cells which inhibit hepatocellular carcinoma growth in vitro and in vivo

α-fetoprotein (AFP), a tumor-associated antigen for hepatocellular carcinoma (HCC), is an established biomarker for HCC. In this study, we created a lentivirus expressing the AFP antigen and investigated the anti-tumor activity of AFP-specific CD8+ T cells, with and without CD4+ T cells, which were activated by either AFP peptide-pulsed or Lenti-AFP-engineered Dendritic cells (DCs) in vitro and in vivo. AFP-specific T cells could efficiently kill HepG2 HCC cells, and produced IL-2, IFN-γ, TNF-α, perforin and granzyme B, with minimal production of IL-10 (a negative regulator of T cell activation). Both strategies activated AFP-specific T cells, but the lentiviral strategy was superior by several measures. Data also support an impact of CD4+ T cells in supporting anti-tumor activity. In vivo studies in a xenograft HCC tumor model also showed that AFP-specific T cells could markedly suppress HCC tumor formation and morbidity in tumor-bearing nude mice, as well as regulate serum levels of related cytokines and anti-tumor molecules. In parallel with human in vitro T cell cultures, the in vivo model demonstrated superior anti-tumor effects and Th1-skewing with Lenti-AFP-DCs. This study supports the superiority of a full-length antigen lentivirus-based DCs vaccine strategy over peptides, and provides new insight into the design of DCs-based vaccines.     

肝干细胞与肝癌干细胞

正常肝干细胞在致癌因素作用下发生基因突变，获得无限增殖能力成为肿瘤干细胞，肿瘤干细胞分化成各种分化等级、表型不一的肿瘤。在正常肝干细胞转化成肝癌干细胞的过程中保留了一些干细胞表型特征和进一步向相对成熟细胞分化的能力。正常肝干细胞是肝癌重要起源。     

人树突状细胞与肝癌细胞系HLE融合细胞的构建

目的构建人源树突状细胞（DC）与肝癌细胞系HLE的融合细胞。方法含15％FCS的RPMI 1640培养HLE细胞,用GM—CSF和IL-4培养成人外周血单核细胞7d,并用TNF-α和PGE2促成熟2d后获得成熟DC;用荧光染料PKH67-GL（绿色荧光）和PKH26-GL（红色荧光）分别标记DC和HLE细胞,以50％聚乙二醇和10％二甲基亚砜为融合剂,构建可供流式细胞仪快速筛选的融合细胞。结果成功构建具有红、绿双色荧光的人源DC与HLE的融合细胞,融合率为16．8％。结论利用PKH67-GL和PKH26-GL为标记,可获得便于快速识别和筛选的DC与肝癌细胞的融合细胞,为使用融合DC疫苗治疗肝癌奠定了基础。