抗肿瘤生物制品项目——恶性肿瘤基因治疗

基因治疗是当今肿瘤治疗研究的最前沿，它为从基因水平上根治癌症开辟了全新途径。随着分子生物学技术的迅速发展，多种淋巴因子的基因已在体外获得克隆及高效表达，重组肿瘤坏死因子(TNF)已应用于人体肿瘤过继免疫治疗。然而，由于TNF半衰期短，迫使应用大剂量冲击，结果导致机体难以耐受的毒副作用，严重影响疗效。     

SLE患者TNF B—2等位基因频率分析

目的 检测系统性红斑狼疮（SLE）患者肿瘤坏死因子（TNF B－2）等位基因频率,寻找SLE的易感基因。方法 采用聚合酶链式反应－限制性片段长度多态性（PCR－RFLP）分析法。结果 SLE患者TNF B－2等位基因频率比正常显著增高（P＜0．01）。结论 TNF B－2等位基因可能是SLE的易感基因或易感基因的标记基因。     

TNF与IFN协同诱导癌细胞凋亡机制的研究进展

综述了肿瘤坏死因子(TNF)与干扰素(IFN)协同诱导肿瘤细胞凋亡的机制.分别从受体、细胞周期、相关基因及凋亡途径4个方面进行阐述,以期为细胞因子的游离及固定药物的机制研究提供理论参考,并对肿瘤细胞因子治疗的临床用药有指导意义.     

人新型肿瘤坏死因子在E.coli中的高效表达、纯化及诱导凋亡活性

采用PCR技术获得了新型的人肿瘤坏死因子基因，在153位氨基酸处引入突变，使Gly突变为Leu.将突变体hTNF基因克隆到表达载体pQE30中，SDS-PAGE结果显示经IPTG诱导后,hTNF基因获得大量表达，通过Ni金属螯合层析柱亲和层析获得到纯化的hTNF融合蛋白，Western blot结果表明hTNF蛋白可与抗TNF的抗血清知识性发生特异性反应，荧光染色实验检测了hTNF蛋白诱导细胞发生凋亡的活性。     

牦牛肥胖基因和肿瘤坏死因子α基因的序列分析

肥胖基因(ob gene)是近年来刚被克隆的新基因，该基因的表达产物瘦蛋白(Leptin)具有调节体重、影响动物生长和繁殖率等一系列生物学功能．肿瘤坏死因子-α(Tumor Necrosis Factor-α，TNF-α)是一种主要由活化单核巨噬细胞和T淋巴细胞产生的多效性细胞因子，时TNF-α基因的基础及应用研究已成为近年来动物抗病育种的研究热点之一，本文克隆及测序分析了牦牛上述两个基因的部分片段。结果表明：ob基因大小为1773bp，TNF-α基因为1160bp，通过genebank中的blastn比较分析牦牛ob基因和TNF—α基因与普通牛种的相应基因同源性都高达98％。     

基因表达系列分析在肿瘤研究中的应用

基因表达系列分析(serial analysis of gene expression, SAGE)是一种快速分析基因表达信息的技术.它不但能快速、详细地分析成千上万个基因,还能发现新基因,因此是基因表达定性和定量研究的一种新的有效手段.近年来此技术广泛应用于肿瘤的研究,了解肿瘤发病机制,识别诊断和治疗肿瘤的新基因.可以预测SAGE在肿瘤研究和诊断过程中的应用将会对肿瘤的认识和治疗产生深远的影响.     

肿瘤坏死因子与临床检测

肿瘤坏死因子(Tumor Necrosis Factor,TNF)最早是从巨噬细胞中发现的仅杀伤癌细胞而不损伤正常细胞的因子,它能激活中性粒细胞并刺激其产生超氧化物,释放溶酶体酶,从而提高杀灭微生物的活性.TNF水平测定对于某些疾病的诊断、判断预后及指导用药均具有重要意义.本文就这几方面国内外研究近况综述如下:一、TNF来源、特点、生理作用TNF-α最早由Carswell等于1975年首先报道的,1984年Penica等分离表达了人的TNF-α的cDNA.目前,人们根据TNT的来源暂分为三种:一是由活化的单核或巨噬细胞分泌产生的称为TNF-α;二是由活化的T-淋巴细胞分泌产生的称为TNF-β;三是由NK细胞分泌产生的称为TNF-γ.人TNF-α是由3个分子量为17KD的单体通过1个二硫键非共价组成的,含有157个氨基酸的非糖蛋白;人TNF-β则是由171个氨基酸组成的、分子量为25KD的糖蛋白,分子内无半胱氨酸.TNF的生物学效应是:适量的TNF可调节机体的免疫及代谢功能,增强机体对入侵病原体的抵抗力,对维持机体内部的稳定状态及抵制各种致病因子的侵袭具有重要意义.但是,TNF的过量表达则与机体的炎症、感染、机体损伤等病理状态有关.TNF-α诱导的各种细胞反应是通过与细胞表面两类特异性受体相互作用而产生的,即55KD的TNF受体(TNF receptor,TNF R_1)和75KD     

活性氧在肿瘤坏死因子信号传导中的作用

肿瘤坏死因子α(TNF-α)是最强的细胞内信号诱导剂之一。它通过对半胱氨酸蛋白酶家族(caspases)、丝裂原激活的蛋白激酶(MAPK)、c-jun N端激酶(JNK)、核转录因子AP-1和NF-κB的活化,可诱导细胞凋亡、分化和基因转录。此外,TNF也可通过调节细胞的还原状态,特别是内源性活性氧的改变来介导细胞内的信号。综述了ROS在TNF所诱导细胞内信号传导的相关内容,以阐述ROS介导TNF在细胞内信号传导及调节细胞的存活和凋亡中的作用。     

肿瘤基因治疗的现状与前景

继肿瘤生物治疗和肿瘤发生分子机制取得重要进展之后,肿瘤基因治疗开始进入基因治疗领域,初步的临床试验结果已显示出肿瘤基因治疗在晚期肿瘤治疗中的优越性及其广阔的临床应用潜能。本文系统论述肿瘤基因治疗产生的背景、原理、各种导入外源基因方法和目的基因的优缺点及其应用前景,以及由此而产生的社会伦理学问题。     

异种同源基因、分子进化与肿瘤治疗

癌症目前是人类主要死因之一,肿瘤生物治疗(主要是免疫治疗和基因治疗)有望成为继手术、化疗和放疗后肿瘤治疗的第4种模式,具有广阔的应用前景.然而至今仍有很多方面制约着肿瘤免疫基因治疗的发展,有必要探索肿瘤免疫基因治疗的新途径.自然界生物在由低级向高级物种进化的过程中,不同种属的生物之间有相当一部分基因是比较保守的,它们在不同种属的生物之间都呈现出在结构和/或功能上某种程度的相似性,这些基因就是异种同源基因.异种同源基因之间的碱基差别是由突变引起的,这种突变大多是中性突变.异种同源基因在进化过程中所形成的这种细微差别可用来打破免疫耐受、增强免疫原性、诱导肿瘤细胞的自体免疫反应进而达到抗肿瘤的目的.用异种同源基因诱导自体免疫反应进行肿瘤免疫治疗必将为肿瘤治疗开辟一条全新的途径,并为其他疾病的治疗提供新思维.同时对利用包括"人类基因组计划"在内的多种生物基因组计划成果的应用和产业化,对理解在生物进化过程中的基因同源性规律等都具有重要意义.综述了异种同源基因、分子进化与肿瘤免疫基因治疗等问题并开展讨论.     

Cloning and expression in Escherichia coli of the gene for human tumour necrosis factor

Tumour necrosis factor (TNF) was found originally in mouse serum after intravenous injection of bacterial endotoxin into mice primed with viable Mycobacterium bovis, strain Bacillus Calmette-Guerin (BCG). TNF-containing serum from mice is cytotoxic or cytostatic to a number of mouse and human transformed cell lines, but less or not toxic to normal cells in vitro. It causes necrosis of transplantable tumours in mice. TNF also occurs in serum of rat, rabbit and guinea pig. Rabbit TNF has been purified recently to give a single band on SDS-polyacrylamide gel electrophoresis (PAGE). The purified TNF had a relative molecular mass (Mr) 40,000 +/- 5,000 measured by gel filtration, and 17,000 by SDS-PAGE. Its isoelectric point is 5.0 +/- 0.3. The necrotic activity in vivo and the cytotoxicity in vitro are produced by the same substance. The gene encoding TNF has been identified in a human genomic DNA library using as a probe a cloned cDNA encoding a portion of rabbit TNF. The regions of this gene encoding an amino-acid sequence corresponding to mature TNF have been expressed in Escherichia coli and the product of this expression isolated in pure form and shown to produce necrosis of murine tumours in vivo.     

Growth of human breast cancer cells inhibited by a luteinizing hormone-releasing hormone agonist

About one-third of human breast cancers require hormones for their continued growth and endocrine ablation or anti-hormone therapy can cause regression of these tumours. As a consequence, ovariectomy in premenopausal women or administration of an anti-oestrogen (tamoxifen) in postmenopausal women represent major options for treatment of metastatic breast cancer. Alternatively, chronic administration of agonistic analogues of luteinizing hormone-releasing hormone (LHRH) causes regression of mammary tumours in experimental animals, and such treatment has shown promise in a small series of premenopausal women with advanced breast cancer. It has been assumed that these results were achieved by suppressing the pituitary-ovarian axis, as the treatment causes a reduction in circulating levels of gonadal steroids similar to that produced by castration. However, LHRH agonists can exert major effects on tissues other than the pituitary in animals and in the human. Such findings, coupled with reports of LHRH in human breast milk and immunohistochemical evidence for the presence of LHRH-like activity in some human breast tumours, prompted us to test whether LHRH agonists could have direct antitumour effects. We now report major direct effects of LHRH and its agonists on the growth of breast tumour cells in culture.     

Lung cancer: intragenic ERBB2 kinase mutations in tumours

The protein-kinase family is the most frequently mutated gene family found in human cancer and faulty kinase enzymes are being investigated as promising targets for the design of antitumour therapies. We have sequenced the gene encoding the transmembrane protein tyrosine kinase ERBB2 (also known as HER2 or Neu) from 120 primary lung tumours and identified 4% that have mutations within the kinase domain; in the adenocarcinoma subtype of lung cancer, 10% of cases had mutations. ERBB2 inhibitors, which have so far proved to be ineffective in treating lung cancer, should now be clinically re-evaluated in the specific subset of patients with lung cancer whose tumours carry ERBB2 mutations.     

Recombinant sTRAIL induces cell death of tumor cell lines as well as primary cancer cells

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a new member of TNF family. It was reported that TRAIL could induce apoptosis of tumor cells but not normal cells in tissue culture system. To further study the biological activity and potential clinical significance, a recombinant soluble TRAIL (rsTRAIL) has been expressed stably in E. coli after transformation of pET28b vector containing the extracellular domain of TRAIL. The yield of rsTRAIL is approximately as high as 60% of whole bacterial proteins. The rsTRAIL, purified by Ni+ -agarose affinity chromatography, could remarkably trigger apoptosis at the concentrations of 0.1-1 μg/mL in all 7 tumor cell lines tested in vitro. However, this killing activity has not been observed in mouse fibroblast cell line (NIH3T3) as normal control. Further investigation shows that the rsTRAIL could also kill primary tumor cells isolated freshly from patients with cardiac cancer, breast cancer and malignant thymoma, while the normal human peripheral blood lymphocytes are not killed under the same conditions. These results provide new evidence that rsTRAIL could induce apoptosis of tumor cells specifically and it could be a new promising medicine for tumor therapy.     

Xenogeneic homologous genes, molecular evolution and cancer therapy

Cancer is one of the main causes for death of human beings to date, and cancer biotherapy (mainlyimmunotherapy and gene therapy) has become the most promising approach after surgical therapy, radiotherapy andchemotherapy. However, there are still many limitations on cancer immunotherapy and gene therapy; therefore great ef-fort is being made to develop new strategies. It has been known that, in the process of evolution, a number of genes, theso-called xenogeneic homologous genes, are well-conserved and show the structural and/or functional similarity betweenvarious species to some degree. The nucleotide changes between various xenogeneic homologous genes are derived frommutation, and most of them are neutral mutations. Considering that the subtle differences in xenogeneic homologousgenes can break immune tolerance, enhance the immunogenicity and induce autologous immune response so as to elimi-nate tumor cells, we expect that a strategy of inducing autoimmune response using the property of xenogeneic homologousgenes will become a new therapy for cancer. Moreover, this therapy can also be used in the treatment of other diseases,such as autoimmune diseases and AIDS. This article will discuss the xenogeneic homologous genes, molecular evolutionand cancer therapy.     

腺病毒介导人MDA-7/IL-24基因对人鼻咽癌细胞CNE的凋亡作用

mda-7(melanoma differentiaton-associated gene 7,黑色素瘤分化相关基因7),其表达产物又称 IL-24(Interleukin-24,白介素24),现有实验证明其对多种肿瘤细胞有明显的生长抑制和凋亡诱导作用,具有广泛的癌症治疗前景和预期.鼻咽癌作为中国南方多发的癌症,目前少有mda-7对其凋亡作用的研究,本实验利用腺病毒载体(复制缺陷型)构建搭载mda-7的重组病毒Ad-mda7及空病毒Ad-GFP,用HEK 293T细胞对病毒进行扩增,得到的重组病毒用于人鼻咽癌细胞CNE的凋亡研究.Hoechst 33258凋亡染色以及流式细胞仪PI染色检测结果表明:重组病毒Ad-mda7相比空病毒Ad-GFP以及PBS处理组,对CNE细胞有较明显的凋亡诱导作用.     

An endogenous tumour-promoting ligand of the human aryl hydrocarbon receptor

Activation of the aryl hydrocarbon receptor (AHR) by environmental xenobiotic toxic chemicals, for instance 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin), has been implicated in a variety of cellular processes such as embryogenesis, transformation, tumorigenesis and inflammation. But the identity of an endogenous ligand activating the AHR under physiological conditions in the absence of environmental toxic chemicals is still unknown. Here we identify the tryptophan (Trp) catabolite kynurenine (Kyn) as an endogenous ligand of the human AHR that is constitutively generated by human tumour cells via tryptophan-2,3-dioxygenase (TDO), a liver- and neuron-derived Trp-degrading enzyme not yet implicated in cancer biology. TDO-derived Kyn suppresses antitumour immune responses and promotes tumour-cell survival and motility through the AHR in an autocrine/paracrine fashion. The TDO-AHR pathway is active in human brain tumours and is associated with malignant progression and poor survival. Because Kyn is produced during cancer progression and inflammation in the local microenvironment in amounts sufficient for activating the human AHR, these results provide evidence for a previously unidentified pathophysiological function of the AHR with profound implications for cancer and immune biology.     

Cancer immunotherapy comes of age

Activating the immune system for therapeutic benefit in cancer has long been a goal in immunology and oncology. After decades of disappointment, the tide has finally changed due to the success of recent proof-of-concept clinical trials. Most notable has been the ability of the anti-CTLA4 antibody, ipilimumab, to achieve a significant increase in survival for patients with metastatic melanoma, for which conventional therapies have failed. In the context of advances in the understanding of how tolerance, immunity and immunosuppression regulate antitumour immune responses together with the advent of targeted therapies, these successes suggest that active immunotherapy represents a path to obtain a durable and long-lasting response in cancer patients.