HSP70-肿瘤肽对小鼠肝癌的抗肿瘤免疫效应

目的：观察HSP70肿瘤肽介导的非MHCⅠ限制性抗肿瘤免疫效应。方法：通过主动免疫和继承性免疫保护试验，观察从热休克小鼠肝癌HCaF细胞纯化的HSP70肿瘤肽对HCaF（MHCⅠ类分子表达阴性）的抗肿瘤免疫效果。结果：用源自HCaF细胞的HSP70肿瘤肽主动免疫小鼠，可获得对HCaF细胞攻击的主动免疫保护效果，雌性组明显优于雄性组。转输HSP70肿瘤肽免疫后抗HCaF细胞攻击的小鼠脾细胞，能产生良好的过继性免疫保护效果，可耐受HCaF细胞反复攻击，以1×107个活瘤细胞攻击亦未致瘤。转输HSP70肿瘤肽体外致敏的脾细胞，也能使宿主产生一定的抗HCaF免疫效果。结论：从HCaF细胞纯化的HSP70肿瘤肽可激发宿主产生非MHCⅠ限制性抗肿瘤免疫保护效应，其强度可藉反复攻击而增强，并可通过免疫脾细胞继承性传递。     

HSPs/肽复合物疫苗免疫治疗小鼠肉瘤的初步实验观察

目的:观察热休克蛋白(heat shock pro-teins HSPs)/肽复合物对Balb/C小鼠S180移植性肉瘤模型免疫治疗的不同效果。方法:两种不同剂量的HSPs/肽复合物疫苗分别对Balb/C小鼠S180肉瘤模型进行预防性的免疫治疗实验观察。结果:混合HSPs/肽复合物蛋白组(含有HSP60、70和GP94)和纯化HSP70/肽蛋白组均可以明显降低成瘤率及抑制肿瘤生长速度、延长小鼠生存时间;部分小鼠甚至可以观察到对生长瘤体的免疫排斥过程,达到完全无瘤健康长期生存。结论:1)HSP70/肽、混合HSPs/肽在小鼠移植性S180肉瘤模型中有良好的免疫预防效果,以混合HSPs/肽效果最好。2)只经过简单纯化后的混合HSPs/肽复合物蛋白具有能提高蛋白产量、缩短纯化时间、减少对蛋白以及携带抗原多肽的破坏等优点,而且其免疫治疗效果优于纯化的HSP70/肽疫苗,可望在临床有广泛的应用前景。     

榄香烯对肿瘤细胞生物学特性的影响及其瘤苗免疫效应机制的研究

为研究我们创制的榄香烯(elemene,E)复合瘤苗主动免疫抗瘤效应的分子机制及其与热休克蛋白(heat shock protein,HSP)的可能关系,本文通过光镜、电镜和流式细胞术(FCM)以热休克为比照,研究了抗癌中药有效组分榄香烯对小鼠H22腹水型肝癌(H-2~-)和L615白血病(H-2~k)细胞某些生物学特性(生存率、形态结构、坏死、凋亡、细胞周期等)的影响;在此基础上着重观察比较了:1.榄香烯或/和热休克H22和L615瘤苗的主动免疫保护效应.2.榄香烯复合瘤苗诸因素(榄香烯、MMC、戊二醛)与热休克对两种肿瘤细胞膜HSP70和HSP90表达的影响.3.分离纯化榄香烯、MMC诱导的H22肿瘤细胞的HSP70-肽复合物体内验证其主动免疫预防效应.     

热休克蛋白72-甲胎蛋白抗原表位肽复合物抗小鼠肝癌Hepal-6细胞免疫的实验研究

目的:以热休克蛋白72(HSP72)-甲胎蛋白(AFP)抗原表位肽复合物免疫小鼠,研究该复合物针对AFP肿瘤是否具有特异性抗肿瘤免疫.方法:以HSP72-AFP多肽复合物皮下注射免疫昆明小鼠,并分别以AFP多肽和HSP72单独免疫小鼠为对照组.ELISA法检测免疫后小鼠血清IFN-γ水平、MTT法检测各免疫组小鼠淋巴细胞对肝癌Hepal-6细胞的杀伤作用、以小鼠体内瘤负荷实验评价蛋白复合物的免疫效应.结果:HSP72-AFP多肽复合物组小鼠血清IFN-γ水平、淋巴细胞对Hepal-6细胞的杀伤作用均显著高于AFP多肽和HSP72组(P＜0.01).HSP72-AFP多肽复合物组瘤体积也明显小于AFP多肽和HSP72免疫组(P＜0.01).结论:HSP72-AFP多肽复合物疫苗可诱导荷瘤小鼠产生针对AFP肿瘤的特异性细胞免疫,其对瘤细胞的杀伤效应明显优于两者单一的多肽纯化疫苗.表明HSP72-AFP多肽复合物可诱导小鼠产生有效的抗肿瘤免疫.     

NH小鼠S180肉瘤热休克蛋白70的纯化

目的建立热休克蛋白70(heat shock protein 70,HSP70)/肽复合物分离、纯化和鉴定的技术平台.方法NH小鼠接种S180肉瘤细胞,将肉瘤组织热休克处理后经Sephacryl S-200HR分子筛、ConA和ADP柱序列层析进行分离,所得蛋白经电泳及Western blot进行相对分子质量及性质鉴定,用Lowry标准曲线法对各阶段洗脱液进行蛋白定量分析,并计算蛋白得率.结果纯化蛋白经鉴定确系相对分子质量为70×103的HSP70蛋白;每克湿重瘤组织可纯化约50 mg HSP70.结论用上述蛋白层析法可分离获得较纯的HSP70蛋白,为临床实际应用提供基础.     

快速蛋白液相色谱系统分离与纯化小鼠结肠癌细胞热休克蛋白70

目的:应用快速蛋白液相色谱(fast protein liquid chromatography,FPLC)系统,建立分离和纯化BALB/c鼠源性结肠癌细胞(CT26)热休克蛋白70(heat shock protein70,HSP70)的方法。方法:用经43℃热处理12h的CT26细胞制备裂解液,经DEAE-Sepha-rose离子交换层析柱和ADP-Agarose亲和层析柱进行连续分离,所得蛋白经不连续SDS-聚丙烯酰胺电泳及Western-blotting进行蛋白相对分子质量及性质鉴定。结果:纯化蛋白经鉴定为相对分子质量70×103的HSP70。结论:通过FPLC系统可分离纯度较高的HSP70。     

肿瘤热休克蛋白70对几种Th1型细胞因子的诱升作用

目的 :研究肿瘤热休克蛋白 70 (HSP70 )免疫后小鼠体内几种Th1型细胞因子水平的变化 ,探讨肿瘤来源的热休克蛋白 70的抗肿瘤免疫机制。方法 :用肿瘤细胞中提纯的HSP70免疫小鼠 ,ELISA法测定免疫前后体内细胞因子水平的变化 ,并观察其抗移植肿瘤的效果。结果 :HSP70有明显的抗肿瘤作用 ,HSP70免疫后的小鼠外周血中IL 2、TNF α、TNF β、IFN γ水平较对照组明显升高 ,差异具有极显著性 (P <0 0 1) ,IFN γ在升高后 4周后仍未见下降。结论 :HSP70对Th1型细胞因子有明确的诱升作用 ,Th1型细胞因子的升高对于机体产生及维持抗肿瘤免疫效应是HSP70抗肿瘤免疫作用的一个重要方面     

gp96-肽复合物体外诱导脾淋巴细胞及其抗肿瘤效应

目的研究H22肿瘤细胞来源的热休克蛋白gp96-多肽复合物在体外诱导小鼠脾淋巴细胞的特异性的细胞毒性T细胞(CTL)的产生及其抗肿瘤效应。方法利用蛋白提取纯化技术、细胞培养技术、流式细胞术、CCK-8法、免疫荧光技术等。结果经鉴定获得纯化的热休克蛋白;流式细胞仪检测表明,经gp96-肽复合物诱导后的CD8T细胞比例达到近70%,远远高于对照组的35%、26%;该活化的CTL细胞在效靶比为50∶1时的肿瘤杀伤率达72%与对照组相比具有统计学意义;激光共聚焦显微镜观察证实实验诱导组的培养上清能诱导H22肿瘤细胞凋亡的形态学改变。结论肿瘤来源的热休克蛋白gp96-肽复合物能诱导小鼠脾淋巴细胞的CTL反应,该活化的CTL具有特异性抗H22肿瘤细胞的免疫保护作用并能分泌免疫活性物质诱导H22肿瘤细胞凋亡。     

热休克蛋白70与肿瘤免疫的相关性

热休克蛋白70(HSP70)在肿瘤免疫方面的作用是近年研究的热点,对其作用机制亦有大量较深入的研究,并已将HSP70多肽复合物应用于临床。该文主要对HSP70在肿瘤免疫方面的作用及机制研究进行概述。     

热休克蛋白70-甲胎蛋白重组复合物抗瘤免疫的实验研究

目的:构建含有分子伴侣热休克蛋白70(HSP70)和甲胎蛋白(AFP)分子的蛋白复合物,研究此蛋白复合物是否具有针对AFP肿瘤诱导产生特异性CTL反应和保护性效应的能力.方法:戊二醛交联HSP70和AFP构建HSP70-AFP复合物.皮下注射免疫Balb/c纯系小鼠,同时设立单独AFP和HSP70免疫小鼠组作为对照.酶联免疫斑点实验(ELISPOT)和酶联免疫实验(ELISA)分别检测免疫后小鼠脾产生细胞因子IFN-γ的细胞数目和抗AFP抗体的水平.采用小鼠体内肿瘤负荷实验评价重组蛋白复合物的免疫效应.结果:经重组蛋白复合物免疫,ELISPOT和ELISA实验结果表明HSP70-AFP复合物免疫组分泌IFN-γ细胞因子的脾细胞数目和产生AFP的抗体水平显著高于AFP和HSP70免疫组(P<0.01).HSP70-AFP复合物免疫组瘤体积也明显小于AFP和HSP70免疫组(P<0.01).结论:研究证实了HSP70的免疫佐剂效应.实验结果表明重组HSP70-AFP复合物的连续免疫可以在小鼠体内产生有效的抗瘤免疫,HSP70-AFP重组蛋白复合物对于今后肿瘤疫苗的研究可能具有一定的意义.     

Farnesyltransferase inhibitors: antineoplastic properties, mechanisms of action, and clinical prospects

Farnesyltransferase (FTase) inhibitors are among the current wave of molecularly targeted anti-cancer agents being used to attack malignancy in a rational manner. A large body of preclinical data indicates that FTase inhibitors block cancer cell proliferation through both cytostatic and cytotoxic effects. Interestingly, FTase inhibitors have rather limited effects on normal cell function, suggesting that they may target unique aspects of cancer cell pathophysiology. The development of FTase inhibitors was predicated on the discovery that the Ras oncoproteins must be post-translationally modified to transform cells. However, recent work indicates that the anti-neoplastic effects of FTase inhibitors depend on altering the post-translational modifications of non-Ras proteins as well. In particular, a critical target protein that responds to FTase inhibition by blocking tumor cell growth is RhoB, an endosomal Rho protein that functions in receptor trafficking. In this review, we survey the biological foundations for the clinical development of FTase inhibitors, and consider some of the latest mechanistic studies that reveal how these agents affect cellular physiology.     

Targeting tumor vasculature with homing peptides from phage display

Tumor vasculature expresses a number of molecular markers at much lower levels than those seen in the blood vessels of normal tissues, and in some cases, such markers are undetectable. The presence of these markers relates to angiogenesis; the same markers are shared by all blood vessels undergoing angiogenesis. The endothelial cells, pericytes and smooth muscle cells, and the vascular extracellular matrix in angiogenic vessels can each express such markers. Molecularly, they represent vascular growth factor receptors, cell adhesion proteins and their receptors. Screening of phage display libraries for peptides that home to tumor vasculature when injected into mice has recently provided a new tool for analyzing the distinguishing features of tumor vasculature. Tumor-homing peptides isolated in this manner, as well as an antibody against a form of fibronectin expressed in tumor blood vessels, have been found to serve as targeting devices to concentrate drugs and other therapeutic materials to tumors in in vivo models. Such a targeting strategy can therefore potentially improve the efficacy of drugs and reduce their side effects.     

Identification of EBV transforming genes by recombinant EBV technology

Epstein-Barr virus (EBV) is able to infect primary B-lymphocytes but usually does not proceed to replicate more virions. Instead, EBV persists as an incomplete virus and expresses 12 gene products that transform the growth of these cells into continuously proliferating lymphoblastoid cell lines. Because EBV is associated with several human malignancies, there is intense interest in delineating the molecular functions of these EBV gene products in transformation. This review focuses on the recombinant EBV technologies that have been developed to introduce specific mutations into EBV and test the functions of these EBV genes in primary B-lymphocyte growth transformation.     

Matrix metalloproteinases in tumor invasion and metastasis

Extensive work on the mechanisms of tumor invasion and metastasis has identified matrix metalloproteinases (MMPs) as key players in the events that underlie tumor dissemination. Studies using natural and synthetic MMP inhibitors, as well as tumor cells transfected with cDNAs encoding the MMPs characterized thus far have provided compelling evidence that MMP activity can induce or enhance tumor survival, invasion and metastasis. Because of the ability of MMPs to degrade extracellular matrix (ECM) proteins, the principal mechanism whereby MMPs promote tumor development has been thought to be the proteolytic breakdown of tissue barriers to invasion and the associated facilitation of circulating tumor cell extravasation. However, recent evidence stemming from the use of novel experimental approaches indicates that MMPs do not play a major role in the process of extravasation itself. Rather, they appear to promote intravasation (the process of penetrating the circulation following invasion of blood vessels) and regulate the relationship between tumor cells and host tissue stroma subsequent to extravasation. In addition, the discoveries that a growing number of proteolytically active MMPs may localize to the cell surface in association with adhesion receptors, and that MMP substrates include latent cytokines and growth factors, provide a new conceptual framework for the mechanisms whereby MMPs influence tumor behavior.     

New aspects of integrin signaling in cancer

Members of the integrin family of cell adhesion receptors influence several important aspects of cancer cell behavior, including motility and invasiveness, cell growth, and cell survival. Engagement of integrins with extracellular matrix (ECM) proteins can activate members of the Rho-family of small GTPases; conversely, Rho- and Ras-family proteins can influence the ability of integrins to bind their ligands. These events impinge on the control of cell motility, and ultimately on invasive and metastatic behavior. Integrin engagement with ECM also has important effects on cell survival, particularly for cells of epithelial origin. In some cases, specific integrins have selective effects on the efficiency of signal transduction in cell survival pathways.     

Role of LMP1 in immune control of EBV infection

The Epstein-Barr virus (EBV) encoded latent membrane protein (LMP1) plays a crucial role in the long-term persistence of this virus within the cells of the immune system. Not only is this protein critical for the transformation of resting B cells by EBV, it also displays pleiotropic effects on various cellular proteins expressed in the host cell. These include up-regulation of expression of B cell activation antigens, adhesion molecules and various components of the antigen processing pathway. Here we discuss how LMP1 acts like an expression 'switch' which, depending on the stage of EBV infection, manoeuvres various pathways that either modulate the immune system towards or against its survival.     

腹部压块对膈肌运动影响的研究

目的 :研究腹部压块对膈肌运动的影响。方法 :选择拟行立体适形放疗患有肺癌或肝脏肿瘤的患者 2 0例。按治疗体位仰卧于体部立体放疗定位负压袋内 ,待患者呼吸平稳后 ,将灯光野的中心点置于膈顶运动的最低点 ,在膈肌运动至最高位时拍摄照片 ,测量膈肌运动的最大幅度 ;然后 ,将心形腹部压块放置于患者剑突下 ,并用定位框架的腹带交叉固定 ,按压程度以不引起患者呼吸困难或其他不适为标准 ,5min后按上述方法再次测量膈肌运动的最大幅度。结果 :2 0例患者未加腹部压块的运动幅度为0 6 2～ 2 6 7cm ,平均 (1 4± 0 6 4)cm ,加腹部压块后的膈肌运动幅度为 0 2 8～ 2 0 8cm ,平均 (1 0±0 5 5 )cm ,加腹部压块后膈肌运动幅度平均减小 (0 4± 0 34)cm ,P =0 0 0 0。加腹部压块后 90 % (18/2 0 )的患者膈肌运动幅度受到不同程度的限制 ,但有 10 % (2 /2 0 )的患者膈肌运动幅度增加。结论 :腹部压块可使大部分患者膈肌运动的幅度减小 ,但少部分患者例外 ,即腹部压块并不能使所有膈肌周围肿瘤的照射容积减少。建议在制定放射治疗计划前应预先进行测量和评价     

ABCG2在肺癌中表达的定量研究

目的 观察ABCG2在肺癌和癌周肺组织的表达,从量化角度阐明其在肺癌组织中表达的病理学意义.方法 常规石蜡包埋、HE切片确诊,用免疫组化SP法检测ABCG2在肺癌和癌周肺组织的定位和表达,用LeicaQ500MC图像分析系统对其表达强度进行定量分析,并用表达的阳性单位(positive unit PU)反映其表达强度.结果 ABCG2蛋白在肺癌和癌周正常肺组织中的表达主要定位在细胞质和细胞膜.在癌周正常肺组织的支气管和细支气管上皮呈弥漫表达,腺上皮呈灶性表达;肺鳞癌和肺腺癌弥漫或大片表达,肺鳞癌表达的PU值高于肺腺癌(P＜0.001),肺大细胞癌和肺小细胞癌不表达,PU值接近于零.癌周肺组织表达的PU值高于各型肺癌(P＜0.05).ABCG2蛋白表达的PU值在肺癌原发灶和转移灶之间无差别(P＞0.05),且与肺癌患者的性别、年龄、转移和TNM分期未见明显相关性(P＞0.05),与肺癌分化程度有关(P＜0.001).分化程度越高,PU值越高,但高分化肺癌和癌周肺组织的表达PU值差异无显著性(P＞0.05).结论 ABCG2蛋白表达程度与肺癌类型及分化程度具有相关性,可能成为判断其指标之一.     

Telomerase and human tumorigenesis

Human cancer cells, unlike their normal counterparts, have shed the molecular restraints to limited cell growth and are immortal. Exactly how cancer cells manage this at the molecular level is beginning to be understood. Human cells must overcome two barriers to cellular proliferation. The first barrier, referred to as senescence, minimally involves the p53 and Rb tumor-suppressor pathways. Inactivation of these pathways results in some extension of lifespan. However, inactivation of these pathways is insufficient for immortalization. As normal cells undergo repeated rounds of DNA replication, their telomeres shorten due to the inability of traditional DNA polymerases to completely replicate the end of the chromosomal DNA. This shortening continues until the cells reach a second proliferative block referred to as crisis, which is characterized by chromosomal instability, end-to-end fusions, and cell death. Stabilization of the telomeric DNA through either telomerase activation or the activation of the alternative mechanism of telomere maintenance (ALT) is essential if the cells are to survive and proliferate indefinitely. Conversely, loss of telomere stabilization by an already-immortalized cell results in loss of immortality and cell death. Together this indicates that telomere maintenance is a critical component of immortality. In this review we attempt to describe our current understanding of the role of telomere maintenance in senescence, crisis, and tumorigenesis.     

Post-translational regulation of COX2 activity by FYN in prostate cancer cells

While increased COX2 expression and prostaglandin levels are elevated in human cancers, the mechanisms of COX2 regulation at the post-translational level are unknown. Initial observation that COX2 forms adduct with non-receptor tyrosine kinase FYN, prompted us to study FYN-mediated post-translational regulation of COX2. We found that FYN increased COX2 activity in prostate cancer cells DU145, independent of changes in COX2 or COX1 protein expression levels. We report that FYN phosphorylates human COX2 on Tyr 446, and while corresponding phospho-mimetic COX2 mutation promotes COX2 activity, the phosphorylation blocking mutation prevents FYN-mediated increase in COX2 activity.