特异性抗肝癌单链抗体二聚体在毕赤酵母中的表达、纯化及生物学活性鉴定

目的:实现特异性抗肝癌单链抗体二聚体在毕赤酵母中高效表达、纯化并鉴定其生物学活性.方法:构建酵母表达载体pGAPZαA-BDM,转化感受态大肠杆菌DH5α,选择测序正确的阳性克隆进行扩增后,电转化酵母细胞,表达抗体二聚体,对表达抗体进行纯化、浓度检测,并鉴定其对肝癌细胞的结合活性,免疫组织化学检测二聚体对肝癌组织抗原的特异性.结果:测序显示成功构建酵母表达载体pGAPZαA-BDM,表达96 h抗体收获量最大,二聚体表达量为30 mg/L菌液,为大肠杆菌的300倍.抗肝癌单链抗体二聚体BDM与三种肝癌细胞结合,而与正常肝细胞不结合,结合效价为1:128.免疫组织化学显示二聚体与肝癌组织结合的阳性率比肝硬化、胃癌、正常肝组织高,差异有统计学意义.结论:成功制备了高表达量、高特异性、较好的活性及稳定性的抗肝癌单链抗体二聚体BDM,为制备免疫纳米颗粒及开展肝癌的放射免疫诊断和靶向治疗奠定了基础.     

全人源肝癌噬菌体单链抗体基因的克隆及活性分析

从噬菌体单链抗体库中筛选克隆全人源肝癌抗体基因并进行活性鉴定.PCR鉴定阳性重组菌中人肝癌ScFv的插入率,以肝癌细胞SMMC-7721为抗原对所建抗体库进行4轮"吸附-洗脱-扩增"的亲和筛选.将筛选后的ScFv采用ELISA法鉴定其与人肝癌细胞的结合活性.ScFv基因插入率为70%.在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮提高,第4轮为第一轮的381倍.利用噬菌体抗体库技术筛选出了肝癌噬菌体单链抗体,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性.     

人源化抗肝癌双链抗体纳米颗粒对荷人肝癌裸鼠放射免疫显像研究

目的：观察99mTc 标记的人源化抗肝癌双链抗体 BDM3及双链抗体纳米颗粒在荷肝癌动物体内的靶向分布及放射免疫显像,探讨其对肝癌的靶向诊断和治疗的价值。方法使用99mTc 对人源化抗肝癌双链抗体 BDM3及双链抗体纳米颗粒进行标记,并测定标记率,将标记好的抗体及抗体纳米颗粒经腹腔注入荷瘤裸鼠体内,在注射后不同时间点进行放射免疫显像。取各脏器及肿瘤组织,计算肿瘤/非肿瘤比值。结果99mTc 标记的双链抗体BDM3和双链抗体纳米颗粒标记率为91%和92%,显像率均达100%。注射后1 h 肿瘤/血比值为（1.63±0.17）和（3.63±0.21）,4 h 肿瘤/血比值为（3.82±0.24）和（5.63±0.26）,8 h 肿瘤/血比值为（0.48±0.21）和（1.41±0.32）,两组比较,差异均有统计学意义（P〈0.01,P〈0.001和 P〈0.01）,表明同位素标记的双链抗体和双链抗体纳米颗粒均能在肿瘤组织中浓聚,而在非肿瘤组织中无浓聚现象,因而具有明确的靶向性,而抗体纳米颗粒因具有双重靶向作用,其浓聚现象较抗体本身更显著,能进一步增加显像效果。结论99mTc 标记的双链抗体 BDM3及其纳米颗粒对肝癌组织具有很好的亲和力,可作为肝癌诊断和治疗的靶向载体。     

载紫杉醇的聚乳酸-卵磷脂纳米级微泡的制备及其在超声介导下对肝癌细胞生物效应的评价

目的:本文旨在了解在超声介导下载药微泡对肝癌的治疗作用.方法:我们的研究中以聚乳酸(poly lactic acid,PLA)和卵磷脂为药物载体,以紫杉醇为模型药物,通过冷冻干燥技术以及改良的超声复乳-溶剂挥发法制备载紫杉醇的聚乳酸-卵磷脂纳米级微泡,考察主要的制备参数:(1)超声时间对载药微泡理化特性(包括:粒径、形态、载药量、包封率和超声介导下体外释药特性)的影响;(2)卵磷脂含量,优化其制备最佳条件;然后考察其对于人肝癌细胞的细胞毒性,及其在超声介导下对荷瘤小鼠的抑瘤率和治疗效果.结果:在制备初乳和复乳过程中我们发现在超声时间均为100 s、PLA与卵磷脂质量比为250∶50时可以制得较好的聚乳酸-卵磷脂纳米级微泡.其平均粒径为615 nm、内部为空心的纳米级微泡,药物包封率可达90.90%±5.79%,载药率可达到8.26%±0.53%,紫杉醇以无定型状态分布在微泡的壳内;其在体外药物释放具有零级释放、缓释以及在超声介导下加快药物释放的特点.并且在紫杉醇浓度为10μg/mL时HepG2人肝癌细胞的增殖率仅为43.37%±3.23%.结论:相对于单纯的紫杉醇注射剂而言,在超声介导下载药纳米级微泡注射剂可以提高抑瘤率并且还能减少对小鼠的不良反应.     

抗肝癌单链免疫毒素的构建、表达及导向研究

目的 制备高效、低毒的抗肝癌单链免疫毒素。 方法 将人突变型肿瘤坏死因子α(mTNF α)与人源化抗肝癌单链抗体hscFv25基因连接,构建pGEx4T-1/hscFv25-mTNF α融合蛋白原核表达载体,在大肠杆菌中表达并纯化目的蛋白,经western blot鉴定之后,进行荷肝癌(SMMC-7721)裸鼠体内初步抑瘤实验,并对治疗后裸鼠肿瘤组织进行抗TNF α的免疫组织化学染色。 结果 原核表达载体pGEX4T-1/hscFv25 mTNF α融合蛋白的表达量占细菌总蛋白的12％,hscFv25-mTNFα治疗组对荷肝癌裸鼠的有效率为5/5,其中2/5为完全缓解,3/5为部分缓解,疗效明显高于mTNFα对照组(F=8.70,P<0.05),治疗组裸鼠的肝、肺等组织中未见转移性病灶,经hscFv25-mTNFα治疗后的肿瘤组织,呈TNFα弥漫阳性反应,阳性颗粒主要位于瘤细胞的胞质中。 结论 hscFv25-mTNFα是高效、低毒的抗肝癌单链免疫毒素。     

索拉非尼免疫毫微球的制备及其抗肝癌效果的实验研究

目的制备抗人肝癌抗体与索拉非尼偶联免疫毫微球,观察其特性及抗肝癌效果。方法通过异型双功能交联剂SPDP,将抗人肝癌单克隆抗体HAb18与索拉非尼(sorafenib,SAF)人血清白蛋白毫微球[HSA(SAF)-NS]偶联,制成抗人肝癌抗体与索拉非尼偶联免疫毫微球HAb18-HSA(SAF)-NS,使用凝集试验检测其活性,光镜和电镜下观察其与人肝癌细胞株SMMC-7721特异性结合。MTT法检测该免疫毫微球的体外杀伤性。于人肝癌裸鼠模型上分别使用HAb18-HSA(SAF)-NS、HSA(SAF)-NS及SAF,检测三者的肿瘤抑制率。结果 HAb18-HSA(SAF)-NS具有单抗活性,能与肝癌细胞特异结合;其体外杀伤SMMC-7721细胞IC 50值为42.4μg/mL,与HSA(SAF)-NS(368.9μg/mL)及SAF(377.5μg/mL)相比,明显降低;体内肿瘤抑制率比HSA(SAF)-NS及SAF显著增强(P0.001)。结论 HAb18-HSA(SAF)-NS具有免疫活性,对肝癌细胞有主动靶向性,体内外均具有比HSA(SAF)-NS及SAF更强的抗癌效果。     

豆楮方对人肝癌细胞株抑制作用的体外实验研究

目的:研究豆楮方对人肝癌细胞株HepG2、SMMC-7721的生长增殖抑制作用.方法:按文献方法制备豆楮方含药血清,用MTT法观察细胞增殖情况.结果:豆楮方含药血清在体外对人肝癌细胞株HepG2、SMMC-7721均有抑制作用,对HepG2的最高抑制率为91.48%,对SMMC-7721的最高抑制率为89.42%,抑制肝癌细胞的效果优于含5-Fu血清.结论:豆楮方具有抗肝癌作用.     

耦联CD133/ABCG2抗体的荧光磁性纳米微粒的制备与靶向性实验

目的制备耦联抗CD133及ABCG2抗体的荧光Fe3O4纳米微粒,用于肝癌的早期诊断和癌细胞转移后的定位。方法采用共沉淀法制备Fe3O4磁性纳米粒子,在其外面包裹羧基化葡聚糖,然后与带有荧光基团的CD133及ABCG2抗体耦联,制备成一种可以检测CD133和ABCG2双阳性细胞的免疫磁性纳米微粒,继而检测其表征和抗性;体外检测制备的磁性纳米微粒对人SP细胞的靶向性;肝癌SP细胞裸鼠皮下种植制作肝癌模型,尾静脉注射经筛选的纳米微粒,用荧光显微镜观察磁性纳米微粒的体内靶向作用。结果成功制备得到了具有磁性、粒度均匀的磁性纳米微粒,且在体内外均有荧光和对肿瘤干细胞的靶向性。结论成功制备得到了可用于检测CD133及ABCG2双阳性肝癌干细胞的免疫荧光磁性纳米微粒。     

三氧化二砷对肝癌细胞增殖的影响

为探讨三氧化二砷 (As2 O3)对肝癌HepG2细胞的增殖抑制作用 ,应用台盼蓝拒染法、MTT比色法及细胞克隆形成试验 ,研究As2 O3 对肝癌细胞的增殖抑制和细胞毒作用。结果发现As2 O3 能显著抑制肝癌细胞的生长 ,5 μmol/LAs2 O3 抑制程度明显强于 1μmol/L。 5 μmol/L和 1μmol/LAs2 O3 作用后细胞克隆形成率分别为 1.5± 1.2 %和 12 3± 2 .2 % ,明显低于对照组的 30 .0± 4.2 % (P <0 .0 1)。As2 O3 对肝癌细胞有较强的细胞毒作用 ,且呈显著剂量和时间依赖性。其对于肝癌细胞的杀伤率高于 5 氟脲嘧啶 ( 5 FU )和丝裂霉素 (MMC) ,但无显著差异。As2 O3 与 5 FU及MMC存在协同效应 ,联合应用 ,对肝癌细胞的杀伤率明显升高。说明As2 O3 具有明显的抑制肝癌细胞增殖的作用 ,有必要进一步探讨其对肝癌的治疗价值     

端粒酶逆转录酶肽-核酸病毒样颗粒疫苗抗肝癌免疫的初步研究

目的设计人端粒酶逆转录酶（hTERT）的新型病毒样颗粒疫苗,并研究其抗肝癌的作用.方法以阳离子抗原肽为桥梁,将人粒细胞、巨噬细胞集落刺激因子和hTERT克隆入真核双表达载体pTCAE中,再将多肽和核酸疫苗结合于同一疫苗颗粒,转染并鉴定其免疫原性,评价其转染效率,并观察其免疫转基因小鼠后激发特异性细胞毒性T淋巴细胞（CTL）反应的有效性.结果该疫苗可激发特异性CTL反应,并对肝癌细胞具有特异性杀伤活性.结论成功构建了具有特异性CTL杀伤活性的hTERT肽-核酸病毒样颗粒疫苗,为进一步探索其体内抗肝癌免疫作用奠定了基础.     

Advances in hepatocellular carcinoma: Nonalcoholic steatohepatitis-related hepatocellular carcinoma

An increase in the prevalence of obesity and diabetes mellitus has been associated with the rise in nonalcoholic fatty liver disease (NAFLD). Two-thirds of the obese and diabetic populations are estimated to develop NAFLD. Currently, NAFLD is the most common etiology for chronic liver disease globally. The clinical spectrum of NAFLD ranges from simple steatosis, an accumulation of fat greater than 5% of liver weight, to nonalcoholic steatohepatitis (NASH), a more aggressive form with necroinflammation and fibrosis. Among the patients who develop NASH, up to 20% may advance to cirrhosis and are at risk for complications of end-stage liver disease. One of the major complications observed in patients with NASH-related cirrhosis is hepatocellular carcinoma (HCC), which has emerged as the sixth most common cancer and second leading etiology of cancer-related deaths worldwide. The incidence of HCC in the United States alone has tripled over the last three decades. In addition, emerging data are suggesting that a small proportion of patients with NAFLD may be at higher risk for HCC in the absence of cirrhosis - implicating obesity and diabetes mellitus as potential risk factors for HCC.     

Immunotherapy of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) represents the third most common cause of cancer-related death worldwide and efficient treatment options are urgently needed. Based on its pathogenesis, in addition to a number of correlative studies, immunotherapy represents a potential therapeutic option for patients with HCC. However, tumors have also evolved numerous immune escape mechanisms, including the generation of cells with immune suppressor functions, such as Tregs and myeloid-derived suppressor cells. It has been shown that these suppressor cells mask tumor-specific immune responses in patients with HCC. Different immunotherapeutic approaches including peptide- and dendritic cell-based therapies have demonstrated promising results in patients with HCC. However, we propose that any of these immunotherapeutic approaches needs to be combined with a therapy specifically targeting suppressor cells in HCC.     

Prevention of hepatocellular carcinoma

Because of its frequency and grave prognosis, preventing hepatocellular carcinoma is an urgent priority. Prevention should be possible because environmental carcinogens-chronic hepatitis B and C virus infections, dietary exposure to aflatoxins, and iron overload-cause the great majority of these tumors. Chronic hepatitis B virus infection accounts for 55% of global hepatocellular carcinomas and 80% of those in the high-incidence Asia Pacific and sub-Saharan African regions. In these regions the infection that becomes chronic is predominantly acquired very early in life. A safe and effective vaccine against this virus is available and its universal inclusion in the immunization of infants has already resulted in a marked reduction of chronic infection and a 70% decrease in the occurrence of hepatocellular carcinoma in those immunized. Chronic hepatitis C virus infection is the major cause of hepatocellular carcinoma in industrialized countries. The infection is mainly acquired in adulthood and, until a vaccine becomes available, prevention will consist mainly of identifying, counselling, and treating chronically infected individuals, preventing spread of the virus by the use of safe injection practices (particularly in intravenous drug abusers), and screening all donated blood for the presence of the virus. 4.5 billion of the world’s population are exposed to dietary aflatoxins. Prevention involves treating susceptible crops to prevent fungal contamination, and handling the foodstuffs in such a way as to prevent contamination during storage. Iron overload in hereditary hemo-chromatosis can be prevented by repeated venesection and in African dietary iron overload by fermenting the home-brewed beer in iron-free containers.     

Prevention of hepatocellular carcinoma

Prevention is the only realistic approach for reducing mortality rates associated with hepatocellular carcinoma (HCC) worldwide. Vaccination against hepatitis B and screening of blood donations are effective measures of primary prevention. Screening of blood donations has led to a substantial reduction in viral hepatitis transmission among the general population, and in Taiwan vaccination against hepatitis B caused a significant reduction in HCC incidence among infants. Primary prevention also includes approaches that alter epigenetic and genetic changes in hepatocytes, known to increase susceptibility to HCC, as well as treatments slowing progression to cirrhosis. The only evidence that chemoprevention reduces HCC risk is a multicenter randomized prospective study in Asian patients with advanced hepatitis B who received the oral nucleoside analogue lamivudine. Circumstantial evidence suggests that HCC risk is also reduced in patients with chronic hepatitis C who have had a sustained virological response to interferon therapy. HCC is not substantially reduced in patients with hepatitis B treated with interferon and patients with hepatitis C who did not respond to interferon. Secondary prevention, that is, prevention of tumor recurrence after hepatic resection or local ablative therapies, has been pursued with different approaches. Retinoids, hepatic embolization with (131)I lipiodol, and adoptive adjuvant immunotherapy have yielded encouraging results. Other approaches, including those based on interferon alfa or beta, provided inconclusive evidence for secondary prophylaxis of HCC, mainly because of the poor methodologies and scientific background of the studies. Dietary interventions and antiaflatoxin agents might help to prevent HCC in susceptible individuals, but the real efficacy of these approaches is far from being demonstrated.     

Treatment of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumours worldwide. The major etiologies and risk factors for HCC development are well defined and some of the multiple steps involved in hepatocarcinogenesis have been elucidated in recent years. Despite these scientific advances and the implementation of measures for early HCC detection in patients at risk, patient survival has not improved during the last three decades. This is due in part to the advanced stage of the disease at the time of clinical presentation, in part due to the limited therapeutic options. These fall into four main categories: (1) surgical interventions, including tumour resection and liver transplantation, (2) percutaneous interventions, including ethanol injection and radiofrequency thermal ablation, (3) transarterial interventions, including embolisation and chemoembolisation and (4) drugs as well as gene and immune therapies. These therapeutic strategies have been evaluated in part in randomised controlled clinical trials that are the basis for therapeutic recommendations. While surgery and percutaneous as well as transarterial interventions are effective in patients with limited disease (1-3 lesions, < 5 cm in diameter) and compensated underlying liver disease (cirrhosis Child A), at the time of diagnosis more than 80% patients present with multicentric HCC and advanced liver disease or comorbidities that restrict the therapeutic measures to best supportive care. In order to reduce morbidity and mortality from HCC, therefore, early diagnosis and the development of novel systemic therapies for advanced disease, including drugs, gene and immune therapies as well as primary HCC prevention are of paramount importance. Further, secondary HCC prevention after successful therapeutic interventions needs to be improved in order to make an impact on the survival of patients with HCC. New technologies, including gene expression profiling and proteomic analyses, should further elucidate the molecular events underlying HCC development and identify novel diagnostic markers as well as therapeutic and preventive targets.     

Prognosis of hepatocellular carcinoma

The prognosis of patients with hepatocellular carcinoma is related to the stage of the tumor at diagnosis and to the degree of liver function impairment induced either by the tumor itself or by the underlying cirrhosis. Any prognostic prediction should also take into account the potential impact of therapeutic interventions. Only surgical resection, liver transplantation and percutaneous ablation achieve a relatively high rate of complete responses in patients with tumors diagnosed at an early stage and may improve survival. By contrast, patients diagnosed at an advanced stage will receive palliative treatment with unproven survival benefits. Each stage and each treatment have their specific prognostic predictors. Thus, the most accurate prognostic system will have to use a specific model for each strata at which patients may be diagnosed: early, intermediate-advanced and terminal. Patients at an early stage may achieve a 5-year survival rate above 50%, those at intermediate-advanced present a 20-50% survival at 3 years and those at terminal stage die within six months. In addition to predicting prognosis, the staging system should also guide the selection of treatment and this is the major advantage of the classification applied in the Barcelona-Clinic Liver Cancer Group.     

Immunology of hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is primarily a malignancy of the liver, advancing from a damaged, cirrhotic liver to HCC. Globally, HCC is the sixth most prevalent cancer and the third-most prevalent reason for neoplastic disease-related deaths. A diverse array of infiltrating immunocytes regulates the development and progression of HCC, as is the case in many other cancers. An understanding of the various immune components during HCC becomes necessary so that novel therapeutic strategies can be designed to combat the disease. A dysregulated immune system(including changes in the number and/or function of immune cells, cytokine levels, and the expression of inhibitory receptors or their ligands) plays a key role in the development of HCC. Alterations in either the innate or adaptive arm of the immune system and cross-talk between them make the immune system tolerant to tumors, leading to disease progression. In this review, we have discussed the status and roles of various immune effector cells(e.g., dendritic cells, natural killer cells, macrophages, and T cells), their cytokine profile, and the chemokine-receptor axis in promoting or impeding HCC.     

Treatment of hepatocellular carcinoma

Opinion statement Hepatocellular carcinoma is the fifth leading cause of cancer worldwide and its incidence is increasing. Surveillance programs allow doctors to identify patients at early stages of the disease, when the tumor may be curable by radical treatments such as resection, liver transplantation, or local ablation. In the West, these treatments can be applied to 30% to 40% of patients. Resection yields favorable results in patients with single tumors and a well-preserved liver function (5-year survival rate is 60%). Recurrence complicates two thirds of the cases, and there is no effective adjuvant treatment. Liver transplantation is the best treatment for patients with single tumors that are less than 5 cm in diameter and liver failure, or in those presenting with three nodules less than 3 cm, but organ shortage greatly limits its applicability. Long-term survival is expected to be around 50% to 70% at 5 years depending upon the drop-out rate of patients on the waiting list. Chemoembolization and local ablation are the neo-adjuvant treatments applied to patients on the waiting list to prevent tumor progression; no controlled study proving their efficacy has yet been published. In nonsurgical candidates, percutaneous treatments (ethanol injection or radiofrequency ablation) are the best therapeutic approach and improve survival in Child-Pugh A class patients with small tumors that achieve initial complete response (5-year survival rate is 40% to 50%). At more advanced stages, chemoembolization, a technique combining intra-arterial chemotherapy and selected ischemia, has shown to slightly improve survival in a meta-analysis of randomized trials. No survival advantages have been demonstrated with intra-arterial or systemic chemotherapy, hormonal compounds, or radiation. New agents, such as inhibitors of the tyrosine kinase receptors of growth factors and antiangiogenic agents, are currently being tested in phase II/III trials.     

Resection of hepatocellular carcinoma

The majority of hepatocellular carcinoma occurs in patients with liver cirrhosis. Although the tumors are often discovered at an early stage during surveillance of these patients, the underlying cirrhosis renders the surgery more difficult and exposes the patients to higher rates of postoperative morbidity and mortality than occurs in other types of liver surgery. Over the past 20 years surgeons have developed new surgical procedures and techniques to firstly reduce the unnecessary resection of liver parenchyma and to decrease intraoperative blood loss. Better patient selection and understanding of prognostic factors will hopefully result in a further decrease in operative risk and postoperative recurrence. Adjuvant chemotherapy may prove effective in reducing the postoperative recurrence but at this stage surgery still remains as the best treatment for patients with recurrent tumor which is accessible to resection.