四跨膜超家族蛋白CD151促血管生成的研究进展

利用促血管生成因子促进血管生成已成为当前治疗缺血性疾病研究的一个热点。CD151蛋白作为四跨膜超家族蛋白(transmembrane-4 superfamily proteins,TM4SF)的重要成员之一,其在促血管生成方面起着重要的作用。CD151在体外能促进血管内皮细胞的增值、迁移及管状结构的形成,在体内能增加大鼠缺血后肢和缺血心肌区域的微血管数量,促进血管生成。CD151蛋白作为一个新的促血管生成因子日益受到大家的关注。本文就CD151促血管生成的研究进展进行综述。     

The distribution of residues in a polypeptide sequence is a determinant of aggregation optimized by evolution

It has been shown that the propensity of a protein to form amyloid-like fibrils can be predicted with high accuracy from the knowledge of its amino acid sequence. It has also been suggested, however, that some regions of the sequences are more important than others in determining the aggregation process. Here, we have addressed this issue by constructing a set of “sequence scrambled” variants of the first 29 residues of horse heart apomyoglobin (apoMb_1-29), in which the sequence was modified while maintaining the same amino acid composition. The clustering of the most amyloidogenic residues in one region of the sequence was found to cause a marked increase of the elongation rate (k_agg) and a remarkable shortening of the lag phase (t_lag) of the fibril growth, as determined by far-UV circular dichroism and thioflavin T fluorescence. We also show that taking explicitly into consideration the presence of aggregation-promoting regions in the predictive methods results in a quantitative agreement between the theoretical and observed k_agg and t_lag values of the apoMb_1-29 variants. These results, together with a comparison between homologous segments from the family of globins, indicate the existence of a negative selection against the clustering of highly amyloidogenic residues in one or few regions of polypeptide sequences.     

Immunotherapy success in prophylaxis cannot predict therapy: prime-boost vaccination against the 5T4 oncofoetal antigen

We have investigated the tumour therapeutic efficacy of homologous and heterologous prime-boost vaccine strategies against the 5T4 oncofoetal antigen, using both replication defective adenovirus expressing human 5T4 (Ad5T4), and retrovirally transduced DC lines (DCh5T4) in a subcutaneous B16 melanoma model (B16h5T4). In naïve mice we show that all vaccine combinations tested can provide significant tumour growth delay. While DCh5T4/Adh5T4 sequence is the best prophylactic regimen (P > 0.0001), it does not demonstrate any therapeutic efficacy in mice with established tumours. In active therapy the Adh5T4/DCh5T4 vaccination sequence is the best treatment regimen (P = 0.0045). In active therapy, we demonstrate that B16h5T4 tumour growth per se induces Th2 polarising immune responses against 5T4, and the success of subsequent vaccination is dependant on altering the polarizing immune responses from Th2 to Th1. We show that the first immunization with Adh5T4 can condition the mice to induce 5T4 specific Th1 immune responses, which can be sustained and subsequently boosted with DCh5T4. In contrast immunisation with DCh5T4 augments Th2 immune responses, such that a subsequent vaccination with Adh5T4 cannot rescue tumour growth. In this case the depletion of CD25⁺ regulatory cells after tumour challenge but before immunization can restore therapeutic efficacy. This study highlights that all vaccine vectors are not equal at generating TAA immune responses; in tumour bearing mice the capability of different vaccines to activate the most appropriate anti-tumour immune responses is greatly altered compared to what is found in naïve mice.     

Signaling events involved in anti-CD20-induced apoptosis of malignant human B cells

Anti-CD20 monoclonal antibodies have been successfully employed in the clinical treatment of non-Hodgkin's lymphomas in both unmodified and radiolabeled forms. Previous publications have demonstrated that the antitumor effects of unmodified anti-CD20 mAb are mediated by several mechanisms including antibody-dependent cellular cytotoxicity, complement-mediated cell lysis, and induction of apoptosis by CD20 cross-linking. In this report, we demonstrate induction of apoptosis by three anti-CD20 monoclonal antibodies [1F5, anti-B1, and C2B8 (Rituximab)]. The magnitude of apoptosis induction was greater with the chimeric Rituximab antibody than with the murine 1F5 and anti-B1 antibodies. Apoptosis could be enhanced with any of the antibodies by cross-linking with secondary antibodies (or Fc-receptor-bearing accessory cells). The signaling events involved in anti-CD20-induced apoptosis were investigated, including activation of protein tyrosine kinases, increases in intracellular Ca²⁺ concentrations, caspase activation, and cleavage of caspase substrates. Our results indicate that anti-CD20-induced apoptosis can be attenuated by PP1, an inhibitor of protein tyrosine kinases Lck and Fyn, chelators of extracellular or intracellular Ca²⁺, and inhibitors of caspases, suggesting that anti-CD20-induced apoptosis may involve modulation of these signaling molecules. We also demonstrated that varying the expression of Bcl-2 did not affect the magnitude of anti-B1-induced apoptosis, possibly because of the sequestering effects of other Bcl-2 family members, such as Bad. These studies identify several of the signal-transduction events involved in the apoptosis of malignant B cells that transpire following ligation of CD20 by anti-CD20 antibodies in the presence of Fc-receptor-expressing cells or secondary goat anti-(mouse Ig) antibodies and which may contribute to the tumor regressions observed in mouse models and clinical trials. Received: 27 May 1999 / Accepted: 1 October 1999     

HIV感染者/AIDS病人高迁移率族蛋白1的表达及其临床意义

通过检测74例处于不同病期的HIV感染者/AIDS患者和10例健康对照者PBMCs中HMGB1 mRNA的表达水平及其外周血浆HMGB1、TNF-a和IL-2水平,比较各组之间表达水平的差异及其与CD4＋T淋巴细胞的关系.发现HMGB1 mRNA的表达水平及血浆HMGB1含量在AIDS病人组明显高于感染者组和正常对照组（P〈0.05）;AIDS患者经HAART治疗后疗效差组HMGB1 mRNA的表达水平及血浆HMGB1含量也明显高于疗效好组（P〈0.05）;而经HAART治疗后效果好且免疫功能恢复的患者HMGB1 mRNA的表达及血浆HMGB1含量均较治疗前明显下降（P〈0.05）;当CD4＋T细胞数低于200/μL时,血浆HMGB1含量以及PBMCs中HMGB1 mRNA表达水平与CD4＋T细胞数呈负相关.显示HMGB1在HIV/AIDS发病及病情进展过程中可能起重要作用,HMGB1血浆含量及PBMCs中HMGB1 mRNA的表达水平高低与HIV/AIDS患者病情轻重密切相关.     

CD151 regulates HGF-stimulated morphogenesis of human breast cancer cells

We previously demonstrated that CD151 forms a functional complex with c-Met and integrin α3/α6 in human salivary gland cancer cells. In the current study, we investigated the involvement of CD151, c-Met, and integrin α3/α6 in the cellular morphogenesis of human breast cancer cells. Knockdown of CD151, integrin α3, or integrin α6 expression abolished branching morphogenesis. Decreased c-Met expression in these cells led to the formation of rudimentary networks and prevented their conversion. Furthermore, hepatocyte growth factor (HGF) promoted cellular morphogenesis by accelerating network reorganization. Immunoprecipitation revealed a specific association between CD151 and c-Met. The involvement of CD151 and integrin α3/α6 in HGF-dependent signaling was confirmed by the decreased Akt phosphorylation in cells lacking CD151, integrin α3, or integrin α6. Hence, the regulation of CD151 expression might contribute to changes in HGF/c-Met signaling and thereby modulate the phenotypic characteristics of cancer cells.     

PKCθ is required for hemostasis and positive regulation of thrombin-induced platelet aggregation and α-granule secretion

Platelet activation due to vascular injury is essential for hemostatic plug formation, and is mediated by agonists, such as thrombin, which trigger distinct receptor-coupled signaling pathways. Thrombin is a coagulation protease, which activates G protein-coupled protease-activated receptors (PARs) on the surface of platelets. We found that C57BL/6J and BALB/C mice that are deficient in protein kinase C θ (PKCθ), exhibit an impaired hemostasis, and prolonged bleeding following vascular injury. In addition, murine platelets deficient in PKCθ displayed an impaired thrombin-induced platelet activation and aggregation response. Lack of PKCθ also resulted in impaired α-granule secretion, as demonstrated by the low surface expression of CD62P, in thrombin-stimulated platelets. Since PAR4 is the only mouse PAR receptor that delivers thrombin-induced activation signals in platelets, our results suggest that PKCθ is a critical effector molecule in the PAR4-linked signaling pathways and in the regulation of normal hemostasis in mice.     

Inhibition of surgical trauma-enhanced peritoneal dissemination of tumor cells by human catalase derivatives in mice

Surgical trauma, which is inevitably associated with the surgical removal of cancer, has been reported to accelerate tumor metastasis. The close association of reactive oxygen species with the trauma and tumor metastasis supports the possibility of using antioxidants for the inhibition of metastasis. To inhibit surgical trauma-enhanced peritoneal dissemination, human catalase (hCAT) derivatives, i.e., hCAT-nona-arginine peptide (hCAT-R9) and hCAT-albumin-binding peptide (hCAT-ABP), were designed to increase the retention time of the antioxidant enzyme in the abdominal cavity after intraperitoneal administration. Both ¹²⁵I-labeled derivatives showed significantly prolonged retention in the cavity compared to ¹²⁵I-hCAT. Cauterization of the cecum of mice with a hot iron, an experimental model of surgical trauma, induced abdominal adhesions. In addition, cauterization followed by colon26 tumor cell inoculation increased lipid peroxidation in the cecum and mRNA expression of molecules associated with tissue repair/adhesion and inflammation in the peritoneum. hCAT derivatives significantly suppressed the increased mRNA expression. The cauterization also increased the number of tumor cells in the abdominal organs, and the number was significantly reduced by hCAT-R9 or hCAT-ABP. These results indicate that hCAT-R9 and hCAT-ABP, both of which have a long retention time in the peritoneal cavity, can be effective at inhibiting surgery-induced peritoneal metastasis.     

一个用于转染细胞分选的双顺反子载体的构建及其应用

为简化转染细胞的分选过程,构建了一个含有细胞表面标志 CD34 基因的双顺反子载体 p3.1-IRES-CD34. 利用来源于脑心肌炎病毒 (EMCV) 的内部核糖体进入位点 (IRES) ,实现目的基因与 CD34 基因的共同表达 . 将绿色荧光蛋白 (EGFP) 作为目的基因插入载体的多克隆位点,然后转染 NIH-3T3 细胞,通过免疫磁珠分选 (MACS) 方法来分选细胞 . 结果表明：对于转染细胞,均可实现快速分选 ( 瞬时转染细胞约 48 h ,稳定转染 10~15 天 ) ,并且获得较高纯度 (95% 以上 ) 的表达目的基因细胞 .     

应用水动力转染技术使人低密度脂蛋白受体、CD81和浸入诱导蛋白L同时在小鼠体内表达

为建立人低密度脂蛋白受体（LDLR）、CD81和浸入诱导蛋白L（Sip—L）等多个HCV感染相关分子共表达的转基因小鼠,首先分别构建了白蛋白启动子调控的人LDLR、CD81和Sip-L基因的小鼠肝脏组织特异性表达载体,将3种质粒同时采用水动力转染技术导入小鼠体内,FIT—PCR和免疫组化技术检测转入体内基因的表达及持续时间。结果表明,转染8h后即可检测到3种基因在体内转录,人LDLR和CD81在转染1～4d后可在50％-90％的肝细胞中高效表达,7d后检测不到目的基因表达。为了进一步延长转染基因在小鼠体内的表达时间,又分别构建了人LDLR、CD81和Sip—L的染色体整合型表达载体,将它们与噬菌体ФC31的整合酶表达载体同时水动力转染小鼠,3种基因在体内表达时间可持续到转染后25d。以上结果表明建立了多个HCV感染相关分子共表达的转基因小鼠,为确定这些分子能否使小鼠感染HCV奠定了基础。