基于暗视场荧光图像分析的大鼠微血管流速测量系统的建立

将荧光标记的自身红细胞注入SD大鼠体内,在荧光显微镜下观测标记红细胞在大鼠微血管中的流动情况,并通过显微摄像系统将整个过程以视频信号的形式存贮。使用视频采集卡将流速变化过程回放采样,得到暗视场下的荧光图像。然后利用帧图像分离出奇偶场的图像分析方法测定血流速度。在该系统下测量流动小室中荧光小球的流速,得到的测量值与实际值之间的误差小于7%,两者没明显的差异(P>0.05),流速测量的上限为9.6mm/s。并在大鼠微循环障碍研究中,应用此系统得到了血流速度随时间变化的情况。     

Self-assembled biomimetic monolayers using phospholipid-containing disulfides

Several phospholipid-based disulfide molecules were synthesized and attached onto the gold-coated silicon wafer using the self-assembling method. The syntheses of these surface-modifying agents were conducted by introducing bromoethylphosphorate (PBr), phosphorylcholine (PC) or phosphorylethanolamine (PE) groups on the terminals of a dialkyl disulfide. After disulfides adsorption onto gold substrate surfaces, the composition, the film thickness, and the conformational order of self-assembled monolayer surfaces were explored and discussed in detail based on reflection-absorption infrared spectroscopy, contact angle measurement, Auger electron spectroscopy, X-ray photoelectron spectroscopy, and so on. The monolayer having the PBr end group could also be converted to a PC surface by treating with trimethylamine. The model functional surfaces of Au-SC11-PC, -PE, -PBr, -OH or corresponding mixed layers were used to mimic biomembrane surfaces. The monolayer having PC groups was found to reduce fibrinogen adsorption as evaluated from protein adsorption experiments using quartz crystal microbalance. It also showed relatively low platelet adherence compare to the glass, PBr and PE surfaces. The cell viability test also revealed that the PC surface displayed lower cytotoxicity than other surfaces.     

Intestinal and oncocytic variants of pancreatic intraepithelial neoplasia. A morphological and immunohistochemical study

We report 2 previously undescribed morphological variants of pancreatic intraepithelial neoplasia (PanIN). The first variant with an intestinal phenotype was associated with mucinous carcinomas that occurred in the tail of the pancreas of 2 men (60 and 65 years old). The carcinomas lacked the characteristic ovarian-like stroma of mucinous cystic neoplasms observed in female patients and did not show a papillary architecture. Whether they represent mucinous cystadenocarcinomas or mucinous carcinomas that arose from the flat variant of intraductal papillary mucinous neoplasms could not be determined with certainty. Microscopically, the intestinal type of PanIN was composed of pseudostratified columnar cells similar to those of colonic adenomas and showing variable degrees of dysplasia. A significant increase in the MIB-1 labeling index correlated with the severity of dysplasia. In contrast to conventional PanIN, the intestinal variant expressed MUC-2 and was MUC-1 negative. The second type of PanIN had an oncocytic phenotype, coexpressed MUC-2 and MUC-1 mucins, and was associated with intraductal oncocytic papillary carcinomas that showed a similar immunohistochemical mucin profile. Both intestinal and oncocytic types of PanIN expressed DPC4 and lacked p53 reactivity. The anatomical separation of the PanINs from the carcinomas and the gradual progression of cytological and architectural abnormalities in both variants of PanIN argue against ductal spread (cancerization of the ducts). The intestinal and oncocytic variants of PanIN broaden the morphological spectrum of this intraductal lesion. Although their significance is unknown, the possibility that these PanIN variants represent cancer precursors should be considered.     

血管内皮生长因子和E26转录因子在乳腺癌中的表达及意义

目的 试图揭示血管内皮生长因子（VEGF）和E26转录因子（E26 transformation-specificl,ETS-1)在乳腺癌组织中的表达规律,探讨其在血管生成和肿瘤浸润转移中的作用机制。方法 应用原位杂交和免疫组织化学链霉素抗生物系－过氧化物酶复合物法（SP）法,检测48例乳腺癌组织中VEGF和ETS－1的mRNA和蛋白的表达。结果 乳腺癌细胞高表达VEGF mRNA和蛋白,阳性率分别为75％（36／48）,70．8％（34／48）,而血管内皮细胞几乎不表达;ETS－1既表达在乳腺癌细胞,也表达在血管内皮细胞。癌细胞中mRNA和蛋白表达阳性率分别为85．4％（41／48）,79．2％（38／48）;VEGF和ETS－1高表达组的血管密度明显高于低表达组（均P＜0．01）;VEGF和ETS－1的表达与组织学分级和淋巴结转移密切相关,并且高表达组的微血管密度明显高于低表达组（P＜0．01）。结论 VEGF和ETS－1可促进乳腺癌血管形成,同时也促进肿瘤的浸润和转移;检测VEGF和ETS－1的表达可做为乳腺癌恶性度,浸润转移等生物学行为的参考指标。     

Chromosome 13q neocentromeres: molecular cytogenetic characterization of three additional cases and clinical spectrum

We report three new cases of chromosome 13 derived marker chromosomes, found in unrelated patients with dysmorphisms and/or developmental delay. Molecular cytogenetic analysis was performed using fluorescence in situ hybridization (FISH) with chromosome-specific painting probes, alpha satellite probes, and physically mapped probes from chromosome 13q, as well as comparative genomic hybridization (CGH). This analysis demonstrated that these markers consisted of inversion duplications of distal portions of chromosome 13q that have separated from the endogenous chromosome 13 centromere and contain no detectable alpha satellite DNA. The presence of a functional neocentromere on these marker chromosomes was confirmed by immunofluorescence with antibodies to centromere protein-C (CENP-C). The cytogenetic location of a neocentromere in band 13q32 was confirmed by simultaneous FISH with physically mapped YACs from 13q32 and immunofluorescence with anti-CENP-C. The addition of these three new cases brings the total number of described inv dup 13q neocentic chromosomes to 11, representing 21% (11/52) of the current overall total of 52 described cases of human neocentric chromosomes. This higher than expected frequency suggests that chromosome 13q may have an increased propensity for neocentromere formation. The clinical spectrum of all 11 cases is presented, representing a unique collection of polysomy for different portions of chromosome 13q without aneuploidies for additional chromosomal regions. The complexity and variability of the phenotypes seen in these patients does not support a simple reductionist view of phenotype/genotype correlation with polysomy for certain chromosomal regions.     

用电子自旋共振成像观测氮氧自由基的透皮特性

在自行研制的电子自旋共振成像(Electron spin resonance imaging, ESRI)系统上建立了活体皮肤电子自旋共振成像的实验模型.观测了氮氧自由基化合物2,2,6,6-四甲基哌啶-1-氧(2,2,6,6-tetramethyl-1-piperidinyloxy,TEMPO)穿透小鼠皮肤组织的动态过程和浓度分布情况.成像结果显示TEMPO化合物对不同方法处理后的皮肤组织的透皮特性有明显差别.TEMPO水溶液难以穿透皮肤,而溶解在二甲基亚枫(Dimethyl sulfoxide,DMSO)中的脂溶性TEMPO溶液可以渗入并穿透皮肤.皮肤表层的角质蛋白对TEMPO有明显的阻隔性, 而且化合物渗入或透过皮肤组织的量具有较强的时间依赖性.本研究结果提示ESRI技术为动态研究某些特殊结构化合物的透皮特性提供了科学有效的分析手段.     

Remodelling of action potential and intracellular calcium cycling dynamics during subacute myocardial infarction promotes ventricular arrhythmias in Langendorff-perfused rabbit hearts

We hypothesize that remodelling of action potential and intracellular calcium (Ca_i) dynamics in the peri-infarct zone contributes to ventricular arrhythmogenesis in the postmyocardial infarction setting. To test this hypothesis, we performed simultaneous optical mapping of Ca_i and membrane potential ( V _m) in the left ventricle in 15 rabbit hearts with myocardial infarction for 1 week. Ventricular premature beats frequently originated from the peri-infarct zone, and 37% showed elevation of Ca_i prior to V _m depolarization, suggesting reverse excitation–contraction coupling as their aetiology. During electrically induced ventricular fibrillation, the highest dominant frequency was in the peri-infarct zone in 61 of 70 episodes. The site of highest dominant frequency had steeper action potential duration restitution and was more susceptible to pacing-induced Ca_i alternans than sites remote from infarct. Wavebreaks during ventricular fibrillation tended to occur at sites of persistently elevated Ca_i. Infusion of propranolol flattened action potential duration restitution, reduced wavebreaks and converted ventricular fibrillation to ventricular tachycardia. We conclude that in the subacute phase of myocardial infarction, the peri-infarct zone exhibits regions with steep action potential duration restitution slope and unstable Ca_i dynamics. These changes may promote ventricular extrasystoles and increase the incidence of wavebreaks during ventricular fibrillation. Whereas increased tissue heterogeneity after subacute myocardial infarction creates a highly arrhythmogenic substrate, dynamic action potential and Ca_i cycling remodelling also contribute to the initiation and maintenance of ventricular fibrillation in this setting.     

人大肠癌中Ⅳ型胶原酶与基底膜免疫组织化学双重染色研究

目的观察Ⅳ型胶原酶和基底膜在大肠癌生长中的相互关系及Ⅳ型胶原酶的表达与ｐ２１的表达之间的关系。方法应用免疫组化双标记方法，对８６例人大肠癌组织的Ⅳ型胶原酶表达及基底膜的改变作对照研究。结果大肠癌组织中Ⅳ型胶原酶的阳性率为８３．７２％（７３／８６），与对照组癌周正常肠粘膜的阳性率１０％差异有非常显著意义（Ｐ＜０．０１）。同时，双染色显示Ⅳ型胶原酶阳性的癌组织基底膜表现为不连续，出现缺口、片段缺失或完全消失等形态。此外，肠癌中Ⅳ型胶原酶的分布与ｐ２１的表达呈正相关（ｒ＝０．９７４，ｐ＜０．０１）。结论Ⅳ型胶原酶的分泌对肠癌基底膜破坏起重要作用，可以作为判断大肠癌生长特性的辅助指标。     

Delivery of a hammerhead ribozyme specifically down-regulates the production of fibrillin-1 by cultured dermal fibroblasts

The hammerhead ribozyme is a small catalytic RNA molecule. Potential hammerhead ribozymes that possess a catalytic domain and flanking sequence complementary to a target mRNA can cleave in trans at a putative cleavage site within the target molecule. We have investigated the potential of hammerhead ribozymes to down-regulate the product of the fibrillin-1 gene (FBN1). Fibrillin is a 347 kDa glycoprotein that is a major constituent of the elastin-associated microfibrils. Mutations in the FBN1 gene are responsible for Marfan syndrome (MFS), a common systemic disorder of the connective tissue. Many FBN1 mutations responsible for MFS appear to act in a dominant-negative fashion, raising the possibility that reduction of the amount of product from the mutant FBN1 allele might be a valid therapeutic approach for MFS. A trans-acting hammerhead ribozyme (FBN1-RZ1) targeted to the 5' end of the human FBN1 mRNA has been designed and synthesized, and shown to cleave its target efficiently in vitro. FBN1-RZ1 cleavage is magnesium dependent and efficient at both 37 and 50 degrees C. Delivery of the FBN1-RZ1 ribozyme into cultured dermal fibroblasts, by receptor- mediated endocytosis of a ribozyme-transferrin-polylysine complex, specifically reduces both cellular FBN1 mRNA and the deposition of fibrillin in the extracellular matrix. These results suggest that the use of hammerhead ribozymes is a valid approach to the study of fibrillin gene expression and possibly to the development of a therapeutic approach to MFS.      

突变MyD88重组质粒的构建及其抑制绿脓杆菌诱导的A549细胞IL-8表达

构建突变MyD88真核表达质粒(MyD88 DN),转染人呼吸道上皮细胞株A549,探讨其对绿脓杆菌及其分泌产物刺激IL-8表达的影响.结果显示突变MyD88成功构建入pcDNA3.1/zeo真核表达质粒,转染A549细胞后,可降低绿脓杆菌培养上清或活菌刺激诱导的IL-8分泌.提示突变MyD88可阻断绿脓杆菌感染引起的呼吸道上皮细胞IL-8释放,为呼吸道炎症的基因治疗提供了新的靶基因.