川藏香茶菜中双聚对映贝壳杉烯的结构鉴定

从川藏香茶菜的干叶和嫩枝中,分离得到了一个对映贝壳杉烯的双聚体,命名为川藏香茶菜戊素,经光谱解析和化学证明确定了其结构为川藏香茶菜甲素的Diels-Alder双聚体。     

Unique interactions of asialo von Willebrand factor with platelets in platelet-type von Willebrand disease

The present studies demonstrate that platelets from patients with platelet-type von Willebrand disease show specific and saturable binding of asialo von Willebrand factor (AS-vWF) under conditions where such binding is not observed with normal platelets. Although specific binding of 125I-AS-vWF to formalin-fixed normal platelets could not be demonstrated, specific binding to fixed patient platelets was seen with an apparent Kd of 1.3 micrograms/mL and specific maximally bound ligand of 0.40 micrograms/10(8) platelets. Preincubation of patient platelets with the antiglycoprotein Ib (anti-GPIb) monoclonal antibody AS-2 reduced total binding close to the level of computer-estimated nonspecific binding. In contrast, binding was not reduced by preincubation with anti-GPIIb/IIIa monoclonal antibody or with 5 mmol/L EDTA. Under stirring conditions, the binding of AS-vWF to fixed patient platelets was accompanied by a strong agglutination response. AS-vWF- induced agglutination was similarly observed in patient but not normal platelet-rich plasma (PRP) in the presence of 5 mmol/L EDTA. In the absence of EDTA, AS-vWF produced a full aggregation response in patient PRP at concentrations as low as 0.1 microgram/mL in contrast to the 2 to 20 micrograms/mL required by normal PRP. Both thromboxane B2 formation and adenosine triphosphate secretion showed an AS-vWF concentration dependence paralleling the aggregation responses. These studies show that a major difference in the platelets from patients with platelet-type von Willebrand disease is the presence of an exposed, high-affinity binding site associated with GPIb that recognizes AS-vWF.     

特发性脊柱侧凸患者椎旁肌结蛋白和波形蛋白的特征表达

目的:研究表明,结蛋白和波形蛋白可反映肌肉损伤再生的情况,同时结蛋白可反应肌力的大小。比较特发性和先天性脊柱侧凸患者顶椎处凹凸两侧椎旁肌形态和结蛋白及波行蛋白的表达,探讨椎旁肌在特发性脊柱侧凸发生发展中的作用。方法:①选择2005-07/2006-11间南方医科大学南方医院脊柱骨病科收治的部分特发性脊柱侧凸患者12例,Cobb角平均73.8°;先天性脊柱侧凸患者5例,Cobb角平均36.0°;腰椎间盘突出症患者2例和胸腰椎管内占位性病变患者3例作为正常对照。所有患者年龄均在10～20岁。②对脊柱侧凸患者顶椎凹凸两侧及正常对照组患者椎旁肌进行活检,术前均签署知情同意书。③采用光镜和电镜下观察椎旁肌的形态改变;SP法分别对光镜切片的结蛋白和波行蛋白进行免疫组织化学染色,在光镜下观察蛋白表达;应用图像分析系统测量结蛋白免疫组织化学染色后反应椎旁肌蛋白表达强弱的吸光度(A)。结果:22例患者全部进入结果分析。①椎旁肌组织形态:特发性脊柱侧凸患者椎旁肌在光镜及电镜下均有病变,凹侧椎旁肌病变较重,凸侧椎旁肌相对正常。②结蛋白免疫组织化学的阳性表达:结蛋白在各组椎旁肌中均有不同程度的表达,组间比较差别显著(P<0.01)。特发性脊柱侧凸及先天性脊柱侧凸患者椎旁肌在顶椎凸侧比正常组结蛋白表达稍强(0.2727±0.0478,0.2768±0.0372,0.2429±0.0272,P<0.05),特发性脊柱侧凸及先天性脊柱侧凸患者椎旁肌在顶椎凹侧相对于正常组表达减弱(0.1898±0.0258,0.1869±0.0306,0.2429±0.0272,P<0.05)。③波行蛋白免疫组织化学的阳性表达:各组间椎旁肌肌纤维内均无波形蛋白表达。特发性脊柱侧凸与先天性脊柱侧凸患者同侧椎旁肌两组间在形态和结蛋白及波行蛋白表达无明显差别(P>0.05)。结论:特发性脊柱侧凸患者椎旁肌内波形蛋白表达均为阴性,提示椎旁肌不存在再生现象。与先天性脊柱侧凸类似,特发性脊柱侧凸患者椎旁肌结蛋白表达凸侧增强及凹侧减弱为继发性改变。     

咳停片对豚鼠咳嗽反射的抑制作用及析方研究

目的：观察咳停片的镇咳作用，进行初步析方研究。评价该处方的配伍合理性。方法：分复方组（咳停片）、无氯组（不含氯化铵的咳停片）和氯化铵组3组，比较3组对枸橼酸雾化吸入或电刺激喉上神经引起的豚鼠咳嗽反射的抑制作用。结果：复方组和氯化铵组能抑制枸橼酸引起的豚鼠咳嗽反射，复方组的作用强度是氯化铵组的1.32倍，是无氯组4倍以上，无氯组无明显作用。复方组，无氯组能抑制电刺激喉上神经引起的豚鼠咳嗽反射，复方组的作用强度是无氯组的2.94倍。氯化铵组无作用。结论：咳停片处方中剔除氯化铵后镇咳作用减弱，表明氯化铵与处方中的中药成分有协同作用。初步证明了该处方的合理性。     

高强度聚焦超声辐照正常猪胰腺的安全性及可行性

目的:应用高强度聚焦超声辐照正常猪胰腺,观察其对胰腺损伤的病理变化,评价其安全性和可行性。方法:实验于2005-04/2006-08在中国医疗技术北京源德生物医学工程实验室完成。①实验方法:6只杂种猪随机等分为A,B2组。常规3%戊巴比妥静脉麻醉动物,建立人工声窗,应用FEP-BY02型高强度聚焦超声治疗机经体表辐照正常猪胰腺,范围3cm×3cm,t2∶t1≈150ms∶150ms,每点治疗次数60次,焦点声强为800W/cm2,步距4mm、行距4mm、层距5mm。②观察指标:A组动物术后当天解剖取出胰腺。B组动物分别在高强度聚焦超声辐照前、后查血、尿淀粉酶等生化指标,并在术后5d解剖,取出胰腺。胰腺标本用1%TTC染色并送常规病理苏木精-伊红染色。实验中动物处置符合动物伦理学标准。结果:①一般情况变化:所有动物在高强度聚焦超声辐照过程中生命体征平稳,术后恢复良好。②胰腺病理变化:所有标本在辐照区均发现凝固性坏死灶,2组均见细胞呈凝固性坏死,核固缩,胞质空泡化,核膜断裂。B组见细胞坏死彻底,膜性结构崩解消失,交界处见炎性细胞浸润。③生化指标变化:B组动物辐照后前5d血、尿淀粉酶无明显变化。结论:高强度聚焦超声能准确的定位损伤在体猪胰腺,对于猪胰腺的消融是安全可行的,且副作用极少。     

葡萄糖酸锌对大鼠心肌缺血再灌注损伤的保护作用

目的:观察心肌缺血再灌注损伤过程中血清中一氧化氮的浓度,一氧化氮合酶及铜锌-超氧化物歧化酶的活性,分析葡萄糖酸锌对心肌的保护作用及其可能机制。方法:实验于2005-10/2006-05在锦州医学院药理学实验室进行,50只SD大鼠随机分为5组,即假手术组,缺血再灌注模型组,葡萄糖酸锌167,250,357mg/kg剂量组。采用左冠状动脉下穿线,拉紧丝线引起心肌缺血,放松丝线给予再灌注的方法建立心肌缺血再灌注损伤动物模型,假手术组,完成模型全部操作,只穿线不结扎。葡萄糖酸锌167,250,357mg/kg剂量组,分别以相应剂量葡萄糖酸锌(溶于蒸馏水中,2mL)灌胃,每次2mL,上下午各1次,两次灌胃给药间隔时间为2.5h。于末次灌胃1h后行冠状动脉左前降支结扎30min,再灌注60min。缺血再灌注模型组,给予等容量的生理盐水灌胃,其他处理同上。采用硝酸还原酶化学比色法测定血清中一氧化氮浓度及一氧化氮合酶活性,抽提法测定血清中铜锌超氧化物歧化酶活性。结果:50只大鼠进入结果分析。①模型组大鼠血清中一氧化氮浓度、血清中总一氧化氮合酶、诱导型一氧化氮合酶及铜锌超氧化物歧化酶的活性较假手术组升高,结构型一氧化氮合酶的活性降低(P<0.01～0.05)。②葡萄糖酸锌250,357mg/kg组大鼠血清一氧化氮浓度较模型组升高(P<0.05,0.01);葡萄糖酸锌167,250,357mg/kg组大鼠血清中总一氧化氮合酶的活性及血清中结构型一氧化氮合酶的活性较模型组升高(P<0.01,0.05),血清中诱导型一氧化氮合酶的活性降低(P<0.01,P<0.05);葡萄糖酸锌357mg/kg组大鼠血清中铜锌-超氧化物歧化酶的活性升高(P<0.01)。③血清中铜锌-超氧化物歧化酶活性与一氧化氮浓度呈正相关(r=0.92,P<0.01)。结论:葡萄糖酸锌对缺血再灌注心肌具有保护作用,作用途径可能为:①通过降低诱导型一氧化氮合酶的活性,增强结构型一氧化氮合酶的活性而提高一氧化氮水平。②参与构成铜锌-超氧化物歧化酶,清除自由基。     

在基质材料中生长的许旺细胞：存活、黏附、三维生长和迁移特性

目的:将许旺细胞与细胞外基质凝胶、聚乳酸-羟基乙酸共聚物纤维丝复合培养,观察许旺细胞的存活、黏附、三维生长和迁移等生物学行为。方法:实验于2005-02/2006-03在四川大学完成。①实验材料:3～6月龄SD大鼠40只。16～24周流产胎儿由四川大学华西二医院计划生育科提供,产妇及其家属均签署知情同意书,实验经医学伦理委员会批准。聚乳酸-羟基乙酸共聚物(中国科学院成都分院化学所提供);细胞外基质凝胶(Sigma)。②实验方法:将聚乳酸-羟基乙酸共聚物溶解,旋转抽丝法制成直径为20～40μm的聚乳酸-羟基乙酸共聚物纤维丝。无菌条件下取胎儿坐骨神经,去除神经外膜,胰酶消化 差速贴壁法去除成纤维细胞,达80%融合时行BrdU标记。③实验评估:将许旺细胞离心制成0.1～0.2mm3的微粒,接种在细胞外基质凝胶中,观察许旺细胞在凝胶中的存活、生长和移行。将许旺细胞制成1×1010L-1的30%细胞外基质凝胶后,分别滴加在经过胶原、细胞外基质凝胶预处理的聚乳酸-羟基乙酸共聚物纤维丝的一端,观察许旺细胞在纤维丝上的黏附、生长和迁移。将BrdU标记的许旺细胞复合细胞外基质凝胶后,置入聚乳酸纤维管用以修复大鼠坐骨神经缺损,术后3,6周取材,BrdU免疫组化染色观察许旺细胞的存活情况。结果:①许旺细胞在无血清状态下能在细胞外基质凝胶中存活,并分裂、增殖,相互移行形成类似Bungner带状的许旺细胞柱状结构。②许旺细胞能贴附在聚乳酸-羟基乙酸共聚物纤维丝上生长并沿纤维丝移行,形成多层排列的类似Bungner带状的许旺细胞柱,细胞外基质凝胶能显著增加贴附和移行细胞数量,而胶原作用不显著。③许旺细胞和细胞外基质凝胶复合移植,6周后BrdU阳性细胞数明显低于第3周(t=3.71,P<0.01),成活细胞能在细胞外基质凝胶中移行。结论:许旺细胞与细胞外基质凝胶、聚乳酸-羟基乙酸共聚物纤维丝等生物材料复合培养时,能存活、分裂、增殖,相互移行形成类似Bungner带状的细胞柱,并可贴附在纤维丝上生长迁移,该生物学行为对神经组织工程具有重要作用。     

Molecular characterization of a novel form of (A gamma delta beta)zero thalassemia deletion in a Chinese family

We have identified and molecularly characterized a novel deletion in the beta-globin gene cluster that is associated with elevated fetal hemoglobin in the adult. The propositus is a homozygote from the Yunnan province of China. The deletion spans about 90 kb of DNA and removes the A gamma, delta, and beta-globin genes. The 5' breakpoint of the deletion is located about 0.13 kb upstream from the A gamma-globin gene, whereas the 3' breakpoint is located about 66 kb downstream from the beta-globin gene, about 13 kb upstream from the breakpoint of the Chinese (A gamma delta beta)zero-thalassemia. Heterozygotes for this Yunnanese form of (A gamma delta beta)zero-thalassemia express between 9% and 17% of fetal hemoglobin, whereas the homozygote present with a mild anemia (Hb = 10.7 g/dl). Comparison of the sites of 3' breakpoints of the Yunnanese and the Chinese (A gamma delta beta)zero-thalassemia mutants is compatible with the hypothesis that an enhancer element is located between the 3' breakpoints of these two mutants. Juxta-position to the G gamma gene of this element may be responsible for the efficient gamma-gene expression in the Yunnanese mutant.     

The von willebrand factor domain-mediating botrocetin-induced binding to glycoprotein IB lies between Val449 and Lys728

Botrocetin, a component of Bothrops jararaca venom, induces von Willebrand factor (vWF)-dependent platelet agglutination and has been proposed as an alternative agent to ristocetin for evaluating vWF function. However, important differences between the vWF-platelet interactions induced by these two agents have suggested that different regions of vWF and the platelet may be involved in the interactions induced by the two agonists. We have recently demonstrated that binding of vWF to the platelet glycoprotein (GP) Ib receptor, either induced by ristocetin or as occurs spontaneously with asialo-vWF or vWF from IIb von Willebrand disease, is mediated by a domain residing on a 52/48- kilodalton (kD) tryptic fragment of vWF. This fragment extends from amino acid residue Val (449) to Lys (728). We have now found that this 52/48-kD fragment blocks botrocetin-induced binding of vWF to platelets and completely inhibits botrocetin-induced platelet agglutination. These results provide evidence that the vWF domain-mediating, botrocetin-induced platelet agglutination lies within the region delimited by this fragment and is therefore close to or identical with that which mediates ristocetin-induced binding and spontaneous binding of vWF to platelet GPIb. Anti-GPIb monoclonal antibodies also blocked agglutination, which showed that botrocetin, like ristocetin, induces binding of vWF to the GPIb receptor.