Therapeutic angiogenesis of mouse hind limb ischemia by novel peptide activating GRP78 receptor on endothelial cells

Therapeutic angiogenesis emerged as a non-invasive mean of promoting neovascularization in ischemic tissues. We have searched for new molecules that induce angiogenesis by screening a phage display combinatory peptide library on endothelial cells. One of the selected peptides identified by binding to endothelial cells under hypoxic conditions was further studied. The aim of this study was to assess the therapeutic value of this peptide, RoY, in a mouse hind limb ischemia model and to identify it's receptor on endothelial cells.RoY, a 12 amino-acid synthetic peptide, induced in vitro angiogeneic activity under hypoxic conditions by increasing endothelial cell proliferation, migration and tube formation. In order to assess its therapeutic properties in ischemic tissues, a hind limb ischemia model was induced in C57BL mice by a femoral artery excision. A single local intramuscular injection of RoY peptide to the operated limb, significantly restored blood perfusion and alleviated hind limb ischemia as determined by a laser Doppler imager. Increased capillary density in histological sections corroborated these findings. Protein precipitation and mass spectroscopy studies identified GRP78, a heat shock protein, as the peptide-binding membrane receptor that was increased on endothelial cell membranes under hypoxic conditions. This study demonstrates the efficacy of RoY peptide in alleviation of hind limb ischemia. In addition, it provides evidence that GRP78 is an angiogenic receptor on hypoxic endothelial cells.     

Viral NS3 helicase activity is inhibited by peptides reproducing the Arg-rich conserved motif of the enzyme (motif VI)

The NTPase/helicase of Flaviviridae viruses is one of the essential components of their replication complex. The enzyme is defined by the presence of seven highly conserved amino acid motifs. Random screening of numerous hepatitis C virus (HCV) derived peptides, revealed a basic amino acid stretch corresponding to motif VI of the HCV NTPase/helicase (amino acids 1487-1500 of the HCV polyprotein). This peptide inhibited the unwinding activity of the enzyme with an IC(50)=0.2 microM. Peptides corresponding to motif VI of HCV, West Nile virus (WNV) and Japanese encephalitis virus (JEV) were synthesized and tested as inhibitors of NTPase and unwinding reactions mediated by the viral enzymes. Peptides distinguished in regard to their length and structure. Between the peptides tested HCV(1487-1500) reproducing the sequence of motif VI was the most potent inhibitor of helicase activities of investigated enzymes. Other respective peptides were rather modest inhibitors. The examined peptides inhibited the Flaviviridae helicases in the following order of potency: HCV(1487-1500)>WNV(1959-1572)>JEV(1962-1975). Interestingly, the susceptibility of the helicase activity to the inhibition by the peptides was similar and in the row: HCV>WNV>JEV. The inhibition results from binding and blockade of the active site of the enzyme lyes beyond the NTP-binding and hydrolyzing site. The kinetic analyses indicated that the binding of the peptides do not interfere with the NTPase activity of the enzymes. The peptide may serve as effective and selective tool to reduce the virus propagation.     

Evaluation of saliva collection devices for the analysis of steroids, peptides and therapeutic drugs

The recovery of steroids, peptides and therapeutic drugs from commercial saliva collection devices was investigated. Saliva, spiked with defined concentrations of the analytes was applied to the Quantisal, three different Salivettes, and the Saliva-Collection-System to investigate effects of volume, exposure time and temperature on the recovery. Additionally, saliva was collected from healthy subjects with the same devices. It was found that glucocorticoids can be measured very well from samples obtained with the synthetic fiber Salivettes and the Quantisal (80-100%). For androgens, the Quantisal and the Saliva-Collection-System reached recoveries >80%. The Quantisal and polyester Salivette achieved best recoveries (>80%) for peptides. The results for the cotton Salivette were extremely poor for melatonin, insulin or IL-8 (<20%). The results from the spike-recovery experiments were confirmed by samples collected from healthy volunteers. For most therapeutic drugs the synthetic fiber Salivettes achieved best recoveries of 100+/-10%. Longer exposure of saliva on the collection devices must be avoided for most of the analytes, due to their limited stability and increased adsorption. In conclusion, no device is suitable for all of the salivary compounds. Strict pre-analytical precautions must be considered (e.g. immediate processing of the sample) to guarantee reliable analytical results.     

多肽用于肿瘤治疗的研究进展

抗肿瘤多肽能够特异靶向肿瘤组织,具有易于合成、低免疫原性、活性高、不良反应轻和来源广泛等优点.抗肿瘤多肽能够通过作用于机体的免疫系统、直接杀伤肿瘤细胞、抑制肿瘤转移等多种不同的机制治疗肿瘤.     

HLA衍生肽RDP1258对大鼠淋巴细胞增殖活性的影响

目的探讨合成HLA衍生肽RDP1258对T细胞活化、增殖活性的影响。方法采用Fmoc固相化学合成法人工合成RDP1258，MTT法分别进行大鼠淋巴细胞增殖试验、大鼠单向混合淋巴细胞反应（MLR）及人外周血淋巴细胞毒试验。结果合成RDP1258纯度达95％以上，相对分子质量与理论值相符；RDP1258显著抑制刀豆蛋白A（ConA）刺激的淋巴细胞增殖；RDP1258能抑制单向MLR反应体系中应答细胞对刺激细胞的反应能力。结论RDP1258体外能抑制由丝裂原或同种异体抗原引起的大鼠淋巴细胞增殖反应。     

心肌肽素对缺氧-再给氧损伤心肌

目的:研究多肽类物质心肌肽素对缺氧-再给氧损伤心肌细胞的保护作用。方法:复制培养心肌细胞缺氧-再给氧损伤模型,缺氧60min,再给氧30min,观察心肌肽素对细胞超微结构的影响。以ACAS570进行细胞内游离Ca^2＋的定性检测;以荧光染料Fura-2-AM定量测定细胞内游离Ca^2＋浓度;以荧光偏振法测定细胞膜脂质流动性。结果:心肌肽素能使心肌细胞线粒体超微结构的损伤减轻;可剂量依赖性地降低[Ca^2＋]_i和提高细胞膜脂质流动性;可明显减少Ca^2＋伪彩色三维图色彩值。结论:心肌肽素对缺氧-再给氧损伤心肌细胞有明显保护作用,可能与其降低[Ca^2＋]sub>i和提高细胞膜脂质流动性有关。     

人工合成IKVAV多肽对星形胶质细胞增殖和Neurocan基因表达的影响

目的观察人工合成IKVAV多肽对星形胶质细胞增殖及硫酸软骨素蛋白多糖核心蛋白Neurocan基因表达的影响,并探讨IKVAV潜在的促进受损脊髓功能恢复的作用。方法设计IKVAV序列,人工合成IKVAV多肽,将星形胶质细胞接种于1%IKVAV包被的培养板上,分别于培养1 d、3 d、5 d、7 d、9 d后在激光共聚焦显微镜下观察IKVAV多肽和星形胶质细胞的组织相容性,用CCK-8法检测星形胶质细胞增殖情况,用荧光定量PCR法检测星形胶质细胞硫酸软骨素蛋白多糖核心蛋白Neurocan的表达,并与对照组比较,进行统计学分析。结果与对照组相比,实验组培养板上星形胶质细胞总数明显减少,但细胞存活率〉95%。荧光定量PCR结果显示实验组星形胶质细胞Neu-rocan的表达量较对照组显著降低。结论人工合成IKVAV多肽可抑制星形胶质细胞增殖、下调硫酸硫酸软骨素蛋白多糖的表达,从而抑制受损脊髓胶质瘢痕的形成,促进脊髓功能恢复。     

胆源性胰腺炎大鼠胰腺胆囊收缩素受体变化的实验研究

目的　探讨胆源性胰腺炎（biliarypancreatitis,BP）大鼠胰腺胆囊收缩素受体（cholecystokininreceptor,CCK-R）和血浆胆囊收缩素八肽（cholecystokininoctapeptide,CCK-OP）的变化及其之间的关系。　方法　采用放射受体分析法检测36只BP大鼠及36只对照组大鼠胰腺组织膜CCK-R和竞争性酶联免疫法检测其血浆CCK-OP的含量。　结果　胆胰管结扎后3h、6h及24h胰腺组织膜CCK-R的最大结合容量（Bmax）值为179.0±1.1、119.0±1.8、及114.0±1.4fmol/ml;平衡解离常数Kd值为0.26±0.01、0.13±0.07、0.14±0.01nM;6h及24hBmax值和Kd值与对照大鼠存在显著差异(P均＜0.05)。BP大鼠血浆CCK-OP含量各为12.55±2.10、0.81±0.16、0.79±0.24PM/L;与正常大鼠相比,胰腺CCK-R在术后6h及24h期间结合位点数减少,亲合力增大（P＜0.05）;血浆CCK-OP含量仅在术后3h与对照组大鼠差异有显著性意义（P＜0.05）。　结论　BP大鼠发病过程中,胰腺CCK-R及血浆CCK-OP均发生了明显的变化。     

心源性脑卒中诊断中血生化标志物检查的临床应用

目的寻找较为有效可行的血生化标志物应用于脑卒中病因学诊断,从而指导临床卒中亚型（尤其是心源性卒中）的明确诊断和后续进一步治疗与预防的开展。方法收集本院神经内科2008年1月至2008年12月间因急性脑卒中住院患者的临床资料。所有患者均经脑CT、磁共振成像（MRI）、心脑血管超声及其他检查手段确诊,并经TOAST分类标准进行病因学评估鉴定。血样于患者急诊入院后24h内采样,进行如下数种血生化标志物检查：C反应蛋白（CRP）,D-二聚体（D—dimer,DD）,脑钠肽（BNP）（均使用ELISA法测定）。并根据检查结果分析卒中亚型诊断中上述血生化标志物的临床应用价值。结果入选71例急性缺血性脑卒中患者。与其他亚型卒中患者比较,在心源性卒中患者中,检查发现存在较高水平的BNP与DD表达（P〈0．05）。结论使用上述血生化标志物检查,可能成为急性期心源性卒中诊断的一种较为可行有效的诊断方法,并指导其他进一步诊断性检查及后续防治措施的开展。     

噬菌体表面表达随机8肽文库的构建

目的:构建噬菌体表面表达随机8肽文库;方法: 采用人工合成随机寡核苷酸、基因重组技术,噬菌体表面表达技术等,构建该文库;采用多聚酶链式反应技术、核酸杂交技术和测序分析评价鉴定。多聚酶链式反应所采用的上游引物包括载体克隆位点处部分碱基和部分外源基因的碱基;在核酸杂交中设计了噬菌体载体克隆位点互补探针,以更加准确地排除无外源基因插入的克隆;在测序鉴定中,选择了2个混合探针杂交阳性的克隆。 结果:以上证实获得独特克隆为2.1×10⁸;亲和富集实验证实所建噬菌体文库可以稳定扩展。结论:我们成功地构建了噬菌体随机8肽文库,其库容量为2.1×10⁸。