权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Study on mechanism of ischemia-reperfusion injury in ischemic pancreas, and development of its preventive modality and viability assessment method.

缺血性胰腺缺血再灌注损伤机制的研究及其预防模式和活力评估方法的发展。

基本信息

批准号：
12470248
负责人：
TERAOKA Satoshi
金额：
$ 8.77万
依托单位：
TOKYO WOMEN'S MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

The mechanism of ischemia-reperfusion injury and impaired microcirculation in ischemic pancreas was was investigated with respect to the biochemical and structural analysis of red cell membrane and cytoskeletal protein, and red cell rouleau formation, deformability and filerability. And followings were clarified.1.The deformability and membrane stability of red blood cells (RBCs) taken from healthy control (group A), nondiabetic dialysis patients (group B), nonuremic diabetic patients (group C) and diabetic dialysis patients (group D) were investigated using Ektacytometry. No difference in deformability index (DI) of whole blood and RBC was observed between group B and A, while DI of whole blood and RBC in group C and D was depressed as treatment with phenyhydrazine was depressed. The DI of RCG containing oxidized hemoglobin was depressed and its membrane stability was augmented. These findings suggested that the interaction among cytoskeletal red cell proteins would be altered. The fr … More agmentation of RBC was examined with RCGs exposed to shear stress (750dyn/cm^2) and T50, when the DI became 0.5 (50%), was 15.6+/-23sec in group A, 20.25+/-2.63sec in group B, 17.9+/-4.02sec in group C and 16.25+/-1.44sec in group D.2.The filterability of diluted RBCs solution through nickel mesh (pore size=4μm) was studied under the continuous negative pressure at -50mmH_2O. As in group B, C and D, the flow rate-pressure curve was shifted to the right as compared with group A, the filterability of RBCs was depressed in group B, C and D.3.Along with spectrin and actin, cytoskeletal red cell protein 4.1 organizes hexagonal lattice complex (mesh-like network) in the horizontal plane of membrane cytoskeletal structure of RBC, The biochemical and structural study of skeletal red cell protein 4.1 proved the binding site of calmodulin/Ca^<++> to protein 4.1(30kDa domain), at which protein 4.1 was hydrophobically bound to fatty acid in the lipid layer of the surface membrane.4..No difference in the band pattern of cytoskeletal red cell proteins by SDS-PAGE was noticed among 4 groups.5.Anisocytosis was seen by microscopic observation of RBC smear stained with Giemsa in groups B and D, although it was confined to little difference.6.As the percentage of annexin V-positive red cells by Flow cytometry was significantly increased in group D, it was suggested that the appearance of phosphatidyl-serine, normally confined to the inner lipid layer, at the outer lipid layer of red cell surface membrane was increased and that the composition of phospholipids in the lipid layer and the asymmetry of the lipid bilayer of red cell surface membrane were deranged.7.Although RBCs repel each other by the negative charge elicited by sciatic acids at the side chain of glycoprotein (glycophorin etc) on red cell surface membrane, the decrease in siaic acids and the defect of glycophorin caused the decreased negative charge and the aggregation of RBCs. The membrane electronic charge was studied by examining the absorbability of RBCs in each group using anion and cation exchange resin. Among 4 groups, no difference in absorbability of RBCs to anion/cation exchange resin was observed. The treatment with endo-β-galactosidase almost completely removed glycoprotein from red cell surface membrane and RBCs aggregated each other. Additionally the treatment with rectin, bound to glycoprotein on red cell surface membrane specifically, depressed the deformability by the observation using laser diffraction method. These findings implied that rouleau formation of RBCs would be mediated by the reduction or the defect of glycoprotein on red cell surface membrane.In conclusion, the deformability and the filerability of RBCs were depressed in diabetic dialysis patients. The asymmetry of lipid b*layers of red cell surface membrane was deranged by the increase in phosphatidylserine in the outer lipid layer in diabetic dialysis patients. The removal of glycoprotein from red cell surface membrane caused the decreased negative charge of red cell surface membrane and RBCs easily aggregated each other. The exposure to the oxidative stress depressed the deformability of RBCs and altered the interaction of cytoskeletal red cell proteins. Less

本文从红细胞膜和细胞骨架蛋白的生化和结构分析、红细胞的变形性、滤过性、红细胞的变形性和滤过性等方面探讨了缺血再灌注损伤和微循环障碍的机制。1.采用透射电镜技术对正常对照组（A组）、非糖尿病透析组（B组）、非尿毒症糖尿病组（C组）和糖尿病透析组（D组）的红细胞变形性和膜稳定性进行了研究。B组与A组相比，全血和红细胞变形指数（DI）无显著性差异，而C、D组的全血和红细胞变形指数（DI）均因苯肼的作用而降低。含氧化血红蛋白的RCG的DI降低，膜稳定性增加。这些结果表明，细胞骨架红细胞蛋白之间的相互作用将被改变。的fr ...更多信息用RCG检测红细胞在剪切力作用下的凝集情况DI为0.5（50%）时，A组T50为15.6 ± 23秒，B组为20.25 ± 2.63秒，C组为17.9 ± 4.02sec，D组为16.25 ± 1.44sec。（孔径为4μm）在-50mmH_2O的连续负压下进行了研究。与A组相比，B、C、D组的流速-压力曲线右移，B、C、D组的红细胞滤过率降低。3.红细胞骨架蛋白4.1与血影蛋白、肌动蛋白一起组成六方晶格复合体（网状网络）在RBC的膜细胞骨架结构的水平面中，通过对骨骼红细胞蛋白4.1的生化和结构研究，证实了蛋白4.1（30 kDa结构域）与钙调素/Ca^++的结合位点，即蛋白4.1与膜表面脂质层中的脂肪酸疏水结合。红细胞膜膜蛋白电泳图谱在4组间无明显差异; 5.红细胞涂片Giemsa染色镜检发现B组和D组红细胞大小不等，但差异不大; 6.流式细胞仪检测Annexin V阳性红细胞百分率在D组明显增加，提示磷脂酰丝氨酸的出现，红细胞表面膜磷脂层中磷脂的组成和红细胞表面膜脂双层的不对称性发生了紊乱红细胞表面膜上的糖蛋白（glycophorin等），唾液酸的减少和糖蛋白的缺陷导致红细胞负电荷减少和聚集。用阴、阳离子交换树脂测定各组红细胞的吸附能力，研究红细胞膜电荷。4组间红细胞对阴/阳离子交换树脂的吸附能力无差异。用内切-β-半乳糖苷酶处理几乎完全去除红细胞表面膜上的糖蛋白，红细胞相互聚集。此外，用激光衍射法观察到，与红细胞表面膜上的糖蛋白特异性结合的rectin处理降低了变形性。提示红细胞膜糖蛋白的减少或缺失可能是红细胞形成红细胞膜红细胞叠连的重要原因，说明糖尿病透析患者红细胞的变形性和滤过性降低。糖尿病透析患者红细胞表面膜脂质B* 层的不对称性因外脂层中磷脂酰丝氨酸的增加而紊乱。红细胞表面膜糖蛋白的去除导致红细胞表面膜负电荷减少，红细胞容易相互聚集。暴露于氧化应激抑制红细胞的变形性和改变细胞骨架红细胞蛋白的相互作用。少