RGD序列肽修饰的丝素蛋白仿生支架材料对骨髓间充质干细胞黏附、增殖的影响

目的:为了促进种子细胞在韧带组织工程支架材料上的黏附增殖,在丝素薄膜上共价接枝人工合成的具有生物活性的GRGDS序列,并观察RGD多肽修饰的丝素蛋白支架材料对骨髓间充质干细胞的黏附、增殖的影响。方法:实验于2006-07/2007-07在华中科技大学同济医学院协和医院中心实验室和骨科实验室完成。利用市购的白色家蚕蚕丝制备丝素蛋白薄膜,在丝素蛋白支架材料表面接上海吉尔公司合成的RGD多肽,为丝素-RGD组,以未接多肽的丝素蛋白材料和细胞培养板分别作丝素组和阳性对照组。取第3代骨髓间充质干细胞接种到以上材料和培养板上培养4,12 h后,沉淀法定量检测黏附的细胞数检测细胞黏附率;培养1,2,3,4 d后比较材料上的细胞标准化细胞密度来反映细胞的增殖程度;培养24 h后对细胞骨架作免疫荧光染色,在激光共聚焦显微镜下观察两组细胞骨架的组织情况。结果:培养4,12 h时丝素-RGD组细胞黏附率均显著高于丝素组(P<0.01),但与阳性对照组无明显差异(P>0.05)。培养1,2,3,4 d时各组细胞的增殖程度无显著性差异(P>0.05)。培养24 h丝素-RGD组细胞完全铺展开,有粗大的细胞骨架组织,细胞内应力纤维清晰可见,其肌动蛋白的荧光强度显著高于丝素组(P<0.05)。结论:RGD多肽修饰能显著促进骨髓基质细胞在丝素蛋白支架材料上的黏附、铺展,但对细胞增殖无明显促进作用。     

采用蛋白指纹图谱技术筛选大肠癌肝转移特异性相关蛋白

目的:应用表面增强激光解析电离飞行时间质谱技术从大肠癌及大肠癌肝转移患者中筛选出大肠癌肝转移患者血清特异性相关蛋白。方法:实验于2005-07/2006-09分别在南方医院消化中心实验室与解放军第一五○医院实验室完成。应用美国CipherGen公司IMAC3(ImmobilizedMetalAffinityCapture,金属亲和表面)芯片和蛋白芯片仪检测44例大肠癌患者及36例大肠癌肝转移患者血清中的蛋白质相对含量。设定所有血清样本检测的蛋白质相对分子质量区间在1500～20000。利用PBSⅡ型蛋白质芯片阅读仪对IMAC3芯片进行检测,所得到的蛋白质以波谱的形式表示。采用BiomarkerWizard软件对2组血清在相同质荷比的蛋白质含量数据进行方差分析,将分析所得到的含量有显著性差异(P<0.05)的蛋白质建立数据库,导入BiomarkerPattern智能统计分析软件,选择相应条件,对其进行分组统计,从而得到能够正确分组的特异性蛋白标志物并构建大肠癌肝转移的诊断模型。采用酶联免疫法检测相同血清标本中的CEA水平,与构建的诊断模型在大肠癌肝转移诊断中作比较。结果:①44例大肠癌患者与36例大肠癌肝转移患者的血清蛋白质在质荷比为2685.64～11813间有16个蛋白质含量有显著差异。②大肠癌组在质荷比为5909处的蛋白质的相对含量高于大肠癌肝转移组[(30.1±9.6)%,(14.5±10.4)%,P≤0.01]。③其中44例大肠癌患者中有38例患者被正确分组,36例大肠癌肝转移患者被正确识别,准确率为92.5%(74/80),灵敏度和特异性分别为100%(36/36),86.4%(38/44)。结论:表面增强激光解析电离飞行时间质谱技术快速、准确、灵敏度、特异性高,通过蛋白芯片仪发现的特异性相关蛋白,有望成为大肠癌肝转移诊断中有应用价值的临床检测指标。     

Cytomegalovirus infection after autologous bone marrow transplantation with comparison to infection after allogeneic bone marrow transplantation

Cytomegalovirus (CMV) infection was detected in 65 of 143 (45%) autologous bone marrow transplant (BMT) patients. CMV pneumonitis occurred in only 2% of the patients and CMV retinitis occurred in none. Infection occurred in half of the 40 initially seronegative patients and 47% of the 94 initially seropositive patients. Among initially seropositive patients, platelet recovery was slower in infected patients than in those not infected (97 v 35 days median, P = .003), and neutrophil recovery was slightly delayed in infected patients (31 days v 24 days, P = .02). Although the incidence of CMV infection was comparable in autologous and allogeneic BMT patients, CMV pneumonitis was less frequent in autologous BMT patients (2% v 12%, P less than .001). The risk for CMV pneumonitis in autologous BMT patients was comparable with that in allogeneic BMT patients without graft-v-host disease (GVHD) (2% v 6%), but significantly lower than the risk in allogeneic BMT patients with GVHD (2% v 23%, P less than .001).     

Structural changes in the Mn4Ca cluster and the mechanism of photosynthetic water splitting

Photosynthetic water oxidation, where water is oxidized to dioxygen, is a fundamental chemical reaction that sustains the biosphere. This reaction is catalyzed by a Mn₄Ca complex in the photosystem II (PS II) oxygen-evolving complex (OEC): a multiprotein assembly embedded in the thylakoid membranes of green plants, cyanobacteria, and algae. The mechanism of photosynthetic water oxidation by the Mn₄Ca cluster in photosystem II is the subject of much debate, although lacking structural characterization of the catalytic intermediates. Biosynthetically exchanged Ca/Sr-PS II preparations and x-ray spectroscopy, including extended x-ray absorption fine structure (EXAFS), allowed us to monitor Mn–Mn and Ca(Sr)–Mn distances in the four intermediate S states, S₀ through S₃, of the catalytic cycle that couples the one-electron photochemistry occurring at the PS II reaction center with the four-electron water-oxidation chemistry taking place at the Mn₄Ca(Sr) cluster. We have detected significant changes in the structure of the complex, especially in the Mn–Mn and Ca(Sr)–Mn distances, on the S₂-to-S₃ and S₃-to-S₀ transitions. These results implicate the involvement of at least one common bridging oxygen atom between the Mn–Mn and Mn–Ca(Sr) atoms in the O–O bond formation. Because PS II cannot advance beyond the S₂ state in preparations that lack Ca(Sr), these results show that Ca(Sr) is one of the critical components in the mechanism of the enzyme. The results also show that Ca is not just a spectator atom involved in providing a structural framework, but is actively involved in the mechanism of water oxidation and represents a rare example of a catalytically active Ca cofactor.     

Modulation of polymorphonuclear leukocyte function by cetiedil

Cetiedil citrate monohydrate inhibits sickling of red cells and aggregation of platelets. We assessed its ability to attenuate polymorphonuclear leukocyte (PMN) function. PMN aggregation in response to 2 X 10(-7) M formyl-met-leu-phe (FMLP) was inhibited in a dose- dependent fashion by cetiedil concentrations ranging from 60 to 250 microM. Additionally, 125 microM cetiedil inhibited PMN aggregation in response to 2 X 10(-7) M FMLP, 20 ng/ml phorbol myristate acetate (PMA), and 1 X 10(-6) M A23187 by 69% +/- 18%, 72% +/- 20%, and 65% +/- 4%, respectively. Inhibition of FMLP-induced aggregation was provided by only 5 min of incubation of the drug with the cells and was partially reversible. Cell viability was unaffected by exposure of PMN to the drug. Correspondingly, 125 microM cetiedil prevented the translocation of calcium from the PMN membrane as assessed by chlorotetracycline fluorescence. Paralleling the effect of the drug on PMN aggregation, 125 microM cetiedil inhibited release of superoxide by 55% and decreased the number of available 3H-FMLP receptors. However, its effect on release of the primary granule constituent, myeloperoxidase, was minimal (4.5% inhibition), while the effect on release of the specific granule product, lactoferrin (27% inhibition), was modest. These studies indicate that cetiedil affects PMN aggregation and superoxide release to a much greater extent than PMN degranulation. Thus, cetiedil may have potential uses in modulating inflammatory response in vivo.     

Early detection of antibodies against rDNA-produced HIV proteins with a flow cytometric assay

There is evidence that some human immunodeficiency virus (HIV)-infected individuals have prolonged periods of seronegativity. A flow cytometric immunoreactive bead (IRB) assay is described for quantitative, simultaneous, and early detection of antibodies to HIV. Polystyrene beads of four diameters, each size coated with a different HIV recombinant DNA-produced protein (p24, p31, gp41, or gp120), bound anti- HIV antibodies detected with fluorescent antiglobulin. The IRB assay was performed on a panel of blood donor samples, many giving consistently false-positive enzyme immunoassay (EIA) and indeterminant Western blot (WB) results. The IRB assay proved as sensitive and more specific than currently licensed EIA and WB tests. Results on serial samples from eight HIV-infected individuals indicated that quantitation of anti-p24 by IRB assay may be useful in monitoring disease progression. Sequential pre- and post-EIA seroconversion sera from 35 HIV-infected homosexual men were tested by the IRB assay using IgM- and IgG-specific fluorescent probes. All 35 cases were IRB assay positive for at least one rDNA-p either before (17 of 35, 49%) or at the time of EIA positivity. Eleven cases (31%) initially had only IgM anti-HIV, primarily to gp41 (17%). In two individuals, the IgM response was detected at least 18 months before EIA seroconversion. The IRB assay is a widely applicable analytic procedure, potentially useful in pretransfusion anti-HIV screening of blood.     

幽门螺杆菌的治疗有望改善——序贯疗法

幽门螺杆菌（H.pylori）的成功根除需要联合使用几种细菌敏感的抗生素。H.pylori生存于酸性环境中,因此对于酸敏感的抗生素,欲使其发挥最大效用并克服表型耐药的发生,就必需结合抑酸治疗。H.pylori根除失败多由表型耐药或获得性耐药所致。根据结核的治疗经验,除耐药性因素外,根除失败主要与疗程和（或）剂量不足有关。由质子泵抑制剂（PPI）和两种抗生素组成的传统三联疗法以及由铋剂、甲硝唑和四环素（BMT）组成的三联疗法是最常用的方案。