Model for Intravital Microscopic Evaluation of the Effects of Arterial Occlusion-caused Ischemia in Bone

An in vivo model has been developed for chronic observation of the effects of ischemia on cortical bone remodeling and perfused vascularity. Diaphragm occluders were implanted around the right common iliac artery of four rabbits and inflated to produce 10 h of ischemia to the limb. Microcirculation was monitored with intravital microscopy of injected fluorescent microspheres and FITC-Dextran 70 through a bone window, the tibial bone chamber implant (BCI). Bone resorption and apposition in the BCI were indicated with mineralization dyes. Between 2 and 12 h following release of the occluder, secondary ischemia/no-reflow and other evidence of reperfusion injury were observed. Vessel damage was suggested by abnormally high leakage of FITC-D70 from the few vessels perfused during secondary ischemia. In the weeks following occluder release perfused vasculature increased beyond pre-occlusion levels. Net bone resorption reached a maximum when vascularity passed normal levels. In order to further validate the arterial occlusion model for osteonecrosis, techniques for (1) confirming bone death and (2) detecting increased leukocyte adherence to endothelial cells were added. The dead cell stain Ethidium homodimer-1 was used to tag dead osteocytes immediately after occlusion and produced a measure designated osteonecrosis index. To detect leukocytes adhering to vessel walls, carboxyfluorescein diacetate, succinimidyl ester was injected at occluder release. An increase in the number of adherent leukocytes was detected. © 1999 Biomedical Engineering Society. PAC99: 8764Rr, 8717-d, 8719Tt     

外固定架治疗桡骨远端粉碎性骨折

目的总结桡骨远端粉碎性骨折的外固定架治疗疗效。方法从1995年～2003年采用外固定架治疗桡骨远端粉碎性骨折46例,并进行随访。结果治疗后均随访1年,术后按Dienst评分,优良率为83．3％。结论外固定架固定牢靠,可早期活动腕关节,是治疗桡骨远端粉碎性、开放性骨折的有效方法。     

p53基因在RA诱导SH-SY5Y细胞分化后低表达

全反式维甲酸(RA)的抑制肿瘤细胞生长和调控细胞分化的作用是一项重要的研究课题。而p53基因是一个重要的肿瘤抑制基因,在RA的作用中是否涉及到p53的作用尚有待阐明。本文用低浓度的RA(10 μmol/L)作用于SH-SY5Y细胞,每2d换一次液,在作用9 d后,用光学显微镜观察SH-SY5Y细胞的形态学变化,并提取细胞质总RNA进行RT-PCR反应半定量检测p53基因表达。结果显示,SH-SY5Y细胞经RA诱导后,细胞生出较长的突起,具有神经元的形态;而对照组保持成纤维细胞的形态、无明显的形态学变化。RT-PCR半定量显示,在细胞诱导后,p53基因表达明显下调。本文结果提示,RA的作用可能独立于p53基因途径,p53基因的表达变化可能是继发于RA的作用。     

郑氏植物蛋白对乳腺癌及其癌前病变患者PBL的刺激作用及其意义

目的 :研究郑氏植物蛋白 (Zheng’splant protein ,ZPP)对淋巴细胞的刺激作用及其在乳腺癌早期诊断与治疗中的应用价值。方法 :采用3 H TdR掺入法测定人外周血淋巴细胞(PBL) )的增殖反应。用流式细胞仪测定ZPP对T淋巴细胞CD3+ 、CD4 + 和CD8+ 抗原表达的调节作用。结果 :ZPP对乳腺癌及其前期病变患者PBL有较强的刺激作用 (SI分别为3.0 0± 1.4 5和 2 .5 3± 0 .80 ) ,明显高于对正常对照组和乳管上皮轻度增生患者PBL的刺激指数 (SI分别为 1.0 6± 0 .17和1.18± 0 .19) (P <0 .0 1)。而PHA对各组PBL的增殖反应均有不同程度的刺激作用 ,但各组间无明显差异 (P >0 .0 5 ) .经ZPP刺激后的PBL中CD3+ 、CD4 + 和CD8+ 细胞均发生增殖反应 ,其中CD4 + 和CD8+ 者更为明显。结论 :ZPP可选择性刺激乳腺癌及其前期病变患者的PBL中T细胞发生增殖反应 ,此作用有可能用于乳腺癌的早期诊断与治疗中     

ST2作为Th2细胞亚群标志以及其与支气管哮喘的关系的研究

各自主要分泌IFN γ和IL 4的Th1和Th2亚群 ,与临床疾病的关系十分密切。如何从表面标志上加以区分是一项迫切需要解决的问题。ST2是近年来提出的Th2细胞的稳定标志物。本工作在体外成功地诱导人脐带血T细胞向Th1或Th2分化的基础上 ,应用逆转录PCR分析了ST2mRNA的表达特点。证实ST2在人Th2细胞上的选择性表达。为了探索ST2、Th2与支气管哮喘的关系 ,本工作进一步检测了正常人和支气管哮喘患者外周血单个核细胞中 β actin、ST2以及IFN γ和IL 4的mRNA水平。结果显示 :支气管哮喘患者ST2mRNA水平升高 ,IL 4水平也明显升高 ,但IFN γ无变化。这提示ST2作为Th2细胞的标志物 ,有可能成为Th2极化性疾病如哮喘发病机制研究的一个参考性标志 ,至于ST2是否有可能作为治疗的靶分子 ,有待进一步探讨     

黄皮酰胺促钾通道开放

一种新发现的具有促智作用的药物——黄皮酰胺能抑制去甲肾上腺素(NE)或KCl引起的血管平滑肌收缩。本研究旨在应用膜片钳(patch clamp)技术探讨黄皮酰胺对Wistar大鼠尾动脉平滑肌细胞膜钾离子通道的作用。单个平滑肌细胞用酶法分离,以细胞封接方式记录离子通道活动。在细胞池内注入2μM黄皮酰胺后,钾离子通道活动明显增强。用本实验室开发的计算机软件(patch clamp analysis system,Version 1.0)计算分析通道活动的特征参数。     

Expression of interleukin-6 in the cornea in response to infection with different strains of Pseudomonas aeruginosa

Strains of Pseudomonas aeruginosa causing keratitis can be either cytotoxic (6206) or invasive (6294), while a strain (Paer1) causing contact lens-induced acute red eye has been shown to be neither. In situ hybridization was used to examine the location and identity of cells expressing interleukin-6 (IL-6) mRNA in the murine cornea and changes in expression in response to infection with different strains of P. aeruginosa. The number of IL-6-positive cells was determined by image analysis. IL-6 protein levels were measured by an enzyme-linked immunosorbent assay. BALB/c mice were challenged by use of the wounded-cornea model with P. aeruginosa 6294, 6206, or Paer1 (2 x 10(6) CFU). At time intervals up to 24 h, postchallenge corneal tissue was probed for IL-6 mRNA. IL-6 mRNA expression was rapidly elevated in the epithelium in response to strains 6294 and 6206. At the conclusion of the experiments, infiltrating inflammatory cells also stained positively for IL-6 mRNA. In contrast, corneas challenged with strain Paer1 showed significant upregulation of IL-6 mRNA only at 4 h postchallenge. Three distinct patterns of IL-6 mRNA expression in the mouse cornea occur in response to these three ocular isolates of P. aeruginosa. The data obtained for mRNA expression in the cornea for all three strains of P. aeruginosa correlated well with IL-6 protein analysis of whole-eye homogenates. Differences in the cytokine responses to these strains correlate with differences in the pathology associated with each strain and may offer an opportunity to develop strategies for the improved management of ocular inflammation.     

雷公藤红素对人肥大细胞系粘附能力和粘附分子表达的影响

雷公藤红素是雷公藤单体之一.本文研究雷公藤红素对人肥大细胞系HMC-1细胞粘附及粘附分子表达的影响.结果发现:HMC-1细胞能自发地表达β1、β3型整合素和CD44,不表达β2型整合素,具有迅速粘附于鼠尾胶的能力,在粘附过程中细胞由圆形、椭圆形变成梭形、多极形.雷公藤红素与HMC-1细胞在粘附前或粘附后共育均能以剂量和时间依赖方式抑制粘附和粘附过程中细胞形态的变化,并可下调粘附分子的表达.提示:雷公藤红素能抑制HMC-1细胞的粘附,这一作用与其抑制粘附分子表达和细胞形态的改变有关.     

人乳头瘤病毒18型L1-E6、L1-E7嵌合基因表达载体的构建及表达

目的 构建HPV 18 L1－E6，L1－E7嵌合基因的表达载体，并在CHO细胞中表达。方法 克隆HPV18 L1－E6和L1－E7基因，插入中介载体pGEMT-Easy中并测序鉴定。采用PCR定点突变法，突变L1－E6，L1－E7基因序列中与转化作用相关的位点，分别与L1基因连接后插入真核表达载体pVAX1，构建真核表达质粒pVAX-1L1 E6Mxx,L1E7Mxx。用磷酸钙沉淀法，转染CHO细胞，以抗HPV－18L1，抗E6和抗E7特异性单克隆抗体（mAb)做ELISA和免疫细胞化学法检测。结果 ELISA检测显示，转染各种pVAX1-LIE6Mxx-L1E7Mxx融合蛋白表达质粒的细胞提取物的P－N值均＞2．1；免疫细胞化学检测，在胞浆，胞核可见棕黄色颗粒。结论 我建的pVAX1-L1E6Mxx-E7Mxx融合蛋白质表达质粒，可在转当细胞内表达相应的L1－E6Mxx和L1－E7Mxx蛋白，为今后进行DNA疫苗的研究奠定了基础。     

Abeta-degrading endopeptidase,neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.