益气复脉汤对脑缺血再灌注损伤大鼠ET及CGRP影响的实验研究

目的：观察益气复脉汤对脑缺血再灌注模型大鼠血浆内皮素（ET）和降钙素基因相关肽（CGRP）含量变化的影响，探讨大鼠脑缺血再灌注损伤的中医药治疗方法。方法：采用线栓法制备大鼠右侧大脑中动脉脑缺血再灌注损伤模型，分别用益气复脉汤高、低剂量组、尼莫地平水溶液予以治疗，然后采用放射免疫法检测血清ET和CGRP含量。结果：模型组脑缺血再灌注大鼠血浆ET含量明显增高，而CGRP下降，治疗后ET值比对照组下降明显（P〈0．01），CGRP值比对照组增高明显（P〈0．05）。结论：益气复脉汤通过降低ET和增高CGRP，能够对脑缺血再灌注损伤脑起保护作用，其疗效优于尼莫地平。     

三种实验性IgA肾病模型的比较

目的探讨建立一种理想的IgA肾病（IgAN）动物模型方法。方法分别采用葡聚糖G200、大肠杆菌外膜蛋白和金葡菌的细胞膜20肽抗原决定簇诱导小鼠IgA肾病模型。用分子生物学和病理学方法对3组IgAN模型小鼠进行鉴定和比较。结果（1）葡聚糖组尿蛋白增高，伴有血尿；免疫荧光显示部分肾小球大量IgA沉积；光镜下肾小球系膜细胞增多，肝和脾可见弥漫性的粉染物质沉积；电镜下肾小球系膜区少量低电子密度的致密沉积物，肝和脾可见淀粉丝样物质沉积。（2）大肠杆菌外膜蛋白组尿蛋白增高，伴有血尿；免疫荧光显示肾小球有少量IgA沉积；光镜下肾小球系膜细胞轻度增多，间质炎细胞浸润明显；电镜下肾小球系膜区无电子致密沉积物。（3）金葡菌细胞膜20肽抗原决定簇组尿蛋白增高，伴有血尿；免疫荧光显示多数肾小球均可见大量IgA沉积；光镜下肾小球系膜细胞增多，伴系膜基质轻度增生；电镜下肾小球系膜区和基底膜的内皮细胞下可见高电子密度的致密沉积物。结论金葡菌细胞膜20肽抗原决定簇组诱导的IgAN模型从临床表现和病理学变化与人IgAN极其相似，是3种IgAN模型中最理想的IgAN模型。     

慢性肾脏病非透析患者脑钠素与动脉粥样硬化及心功能的关系

目的 研究脑钠素（BNP）与慢性肾脏病（CKD）非透析患者动脉粥样硬化及心功能不全的关系。 方法 采用双抗夹心免疫荧光法检测203例CKD非透析患者与16例高血压患者对照组全血BNP水平，分析其与颈动脉超声结果、心脏彩超结果及既往心血管疾病史的关系。 结果 CKD非透析患者BNP水平与对照组相比显著升高[M（范围）：54.40(15.10～ 173.00) ng/L比9.35(7.35～15.00) ng/L，P < 0.01]。Spearman相关分析显示CKD患者BNP与颈动脉内膜中层厚度（IMT）、左室心肌重量指数（LVMI）等呈正相关。存在颈动脉斑块、左室肥厚或既往发生过心血管事件的患者血BNP水平显著增高。多元回归分析显示LVMI、既往心血管事件均是影响BNP水平的独立因素。 结论 CKD非透析患者BNP水平和动脉粥样硬化性疾病、左室肥厚及心功能不全相关，提示BNP水平可作为一项评价CKD非透析患者心功能及动脉粥样硬化的敏感生物学指标。     

RGD-based strategies for selective delivery of therapeutics and imaging agents to the tumour vasculature

During the past decade, RGD-peptides have become a popular tool for the targeting of drugs and imaging agents to alphavbeta3-integrin expressing tumour vasculature. RGD-peptides have been introduced by recombinant means into therapeutic proteins and viruses. Chemical means have been applied to couple RGD-peptides and RGD-mimetics to liposomes, polymers, peptides, small molecule drugs and radiotracers. Some of these products show impressive results in preclinical animal models and a RGD targeted radiotracer has already successfully been tested in humans for the visualization of alphavbeta3-integrin, which demonstrates the feasibility of this approach. This review will summarize the structural requirements for RGD-peptides and RGD-mimetics as ligands for alphavbeta3. We will show how they have been introduced in the various types of constructs by chemical and recombinant techniques. The importance of multivalent RGD-constructs for high affinity binding and internalization will be highlighted. Furthermore the in vitro and in vivo efficacy of RGD-targeted therapeutics and diagnostics reported in recent years will be reviewed.     

Identification of new immunogenic peptides in conserved regions of hepatitis C virus (HCV) 1b with the potentiality to generate cytotoxic T lymphocytes in HCV1b+ HLA-A24+ patients

Aim: Hepatitis C virus (HCV) 1b is resistant to standard interferon therapy and has a high risk of developing into hepatocellular carcinoma at the late stage of infection. Therefore, new therapeutic modalities for HCV1b infection must be developed. One approach would be active specific immunotherapy with highly immunogenic HCV1b peptides. Methods: HCV1b-derived 44 synthetic peptides were selected based on their binding scores to HLA-A24. Peptide-specific IgG were measured by ELISA. Peptide-specific cytotoxic T-lymphocytes (CTLs) were induced in vitro by repeated peptide-stimulation. Results: We identified three novel candidate peptides of HCV1b proteins containing HLA-A24 binding motifs. Each of them had the ability to induce HLA-A24-restricted and peptide-specific CTL activity, and IgGs specific to each of them were detected in the plasma of HCV1b patients. Among these three peptides, a peptide NS5A 2132-2142 was recognized by both cellular and humoral immunities in the majority of blood samples of patients tested. More importantly, the peptide-stimulated peripheral blood mononuclear cells (PBMCs) showed cytotoxicity against cells cotransfected with NS5A and HLA-A2402 genes in an HLA-restricted manner. This is an additional report to our previous study. Conclusion: These findings may provide a new insight into the development of a peptide-based specific immunotherapy for HCV1b-infected patients.     

Proposed diagnostic thresholds for gestational diabetes mellitus according to a 75-g oral glucose tolerance test. Maternal and perinatal outcomes in 3260 Danish women.

AIMS: To study if established diagnostic threshold values for gestational diabetes based on a 75-g, 2-h oral glucose tolerance test can be supported by maternal and perinatal outcomes. METHODS: Historical cohort study of 3260 pregnant women examined for gestational diabetes on the basis of risk indicators. Information on oral glucose tolerance test results and clinical outcomes were collected from medical records. RESULTS: There was an increased risk of delivering a macrosomic infant in women with 2-h capillary blood glucose of 7.8-8.9 mmol/l compared with women with 2-h glucose < 7.8 mmol/l. Despite treatment, 2-h glucose of 9.0-11.0 mmol/l and > or = 11.1 mmol/l were both associated with increased rates of macrosomia, spontaneous preterm delivery, hypertensive complications, and neonatal hypoglycaemia. Adverse outcomes tended to be more frequent in women with 2-h glucose > or = 11.1 mmol/l than in women with 2-h glucose of 9.0-11.0 mmol/l. CONCLUSIONS: The risk for several maternal and perinatal complications increased with the diagnostic threshold for 2-h glucose. Large-scale blinded studies are needed to clarify the question of a clinically meaningful diagnosis of gestational diabetes mellitus. Until these results are available, a 2-h threshold level of 9.0 mmol/l after a 75-g oral glucose tolerance test seems acceptable.     

A cyclic peptide–polymer probe for the detection of Clostridium botulinum neurotoxin serotype A

A Botulinum neurotoxin serotype A (BoNT/A) ELISA detection system was developed based upon an 11-mer cyclic peptide, termed C11-019, that was identified through peptide phage display technology. The assay employs a sandwich format using the C11-019 cyclic peptide attached to a PEMA (poly(ethylene maleic anhydride)) matrix as the capture phase and anti-BoNT/A polyclonal antibodies as the detection phase. Results reported demonstrate that the C11-019 peptide–polymer can specifically bind to BoNT/A with no cross-reactivity to other serotypes examined in assay buffers and a variety of body fluids and foodstuffs. When a highly sensitive chemiluminescent substrate was engaged, the detection of 1 pg/mL could be readily achieved within 3 h with a linear range of 0.1–1 ng/mL. These results demonstrate that an inexpensive peptide–polymer-based capture ELISA system can be used for rapid, sensitive and highly specific BoNT detection.     

应用激光显微分离技术在骨肉瘤组织原位筛选噬菌体肽库

目的将激光显微分离(laser capture microdissection, LCM) 技术应用于噬菌体表面呈现肽库的筛选过程,建立一种可以直接在天然组织中筛选肽库的方法。方法将新鲜人骨肉瘤组织块在噬菌体肽库溶液振荡孵育后制成组织冰冻切片,免疫组化染色检测噬菌体在组织中的浸润扩散。改进常规LCM切片处理方法,以冻干法替代酒精/二甲苯法脱水,以期在LCM操作过程中提高切片上噬菌体的存活率。LCM法分离摄取骨肉瘤切片上的肿瘤靶细胞,转染回收这些细胞上特异结合的噬菌体。滴定法检测所筛选的噬菌体对特异性细胞的亲和力。结果利用LCM技术,可以由肿瘤冰冻切片上收集到足够的与特异性噬菌体短肽,应用于噬菌体表面呈现肽库筛选。经过3轮筛选后所获得的噬菌体与人骨肉瘤组织的特异性亲和力提高16倍。结论本研究首次将LCM技术应用于噬菌体表面呈现肽库的筛选,可以使我们直接在新鲜人肿瘤组织中筛选与特定细胞群甚至单个细胞亲合的短肽;同时又避免了天然组织中其他杂质细胞的污染,为研制细胞特异性导向载体提供了一个新的途径。     

诱生型一氧化氮合酶特异性抑制肽的制备

目的：通过噬菌体展示技术筛选iNOS特异性抑制肽。方法：将iNOSFAD结合区及其附近序列的基因片段装入pET-28A( )，在大肠杆菌BL21中表达，His.bind^TM亲和层析柱纯化目的蛋白，使用纯化蛋白筛选Ph.D.-12^TM噬菌体库，筛选iNOS活性抑制作用较高的噬菌体克隆，测序并合成其中具有一致序列的短肽。结果：得到具有较高表达量的目的蛋白，经His.Bind^TM柱亲和层析纯化后纯度大于95％，以纯化蛋白筛选Ph.D.-12^TM噬菌体库，经4轮筛选获得10株iNOS活性抑制作用较高的噬菌体克隆，测序发现其中5株序列完全相同，合成该12肽，初步研究表明其对iNOS表现为高浓度抑制，低浓度兴奋的作用，而对nNOS及eNOS则没有影响。结论：以iNOSFAD片段蛋白为靶蛋白筛选得到的克隆对iNOS活性具有特异性影响，可根据这些特征设计合成小分子前导药物，创造新的活性药物。     

Effect of amyloid peptides on the increase in TrkA receptor expression induced by nicotine in vitro and in vivo

The ability of nicotine to induce a cytoprotective or neuroprotective action occurs through several down-stream mechanisms. One possibility is that the drug increases the expression of tyrosine kinase A (TrkA) nerve growth factor (NGF) receptors. Certain β-amyloid peptides (e.g., Aβ1–42) have been shown to bind with high affinity to α7 nicotinic receptors and thus interfere with a potentially neurotrophic influence. Treatment of differentiated PC-12 cells with nicotine produced a concentration-dependent increase in cell-surface TrkA receptors that occurred concomitantly with cytoprotection. The effect of nicotine was blocked by either of the α7 receptor antagonists α-bungarotoxin (α-BTX) or methyllycaconatine. The cytoprotective action of nicotine also was inhibited by pretreatment with 10–100 nM Aβ1–42. Nicotine also was administered (four injections of 30 μg, spaced evenly over 24 h) to rats by direct injection into a lateral cerebral ventricle. Brain TrkA expression was increased significantly in hippocampus and entorhinal cortex (up to 32% above control), with no changes found in cerebral cortex or hypothalamus. The nicotine-induced increases in TrKA expression in hippocampus and entorhinal cortex were significantly inhibited by 10 μg α-BTX or by 10 nmol Aβ1–42. Therefore, physiologically relevant concentrations of Aβ1–42 can prevent nicotine-induced TrkA receptor expression in brain regions containing cholinergic neurons susceptible to the neurotoxicity associated with Alzheimer’s disease.