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

从红细胞膜和细胞骨架蛋白的生化和结构分析、红细胞皱纹的形成、变形性和滤过性等方面探讨了缺血胰腺缺血再灌注损伤和微循环障碍的机制。本研究的主要研究内容如下：1.用红细胞电泳仪检测了健康对照组(A组)、非糖尿病透析患者(B组)、非尿毒症糖尿病患者(C组)和糖尿病透析患者(D组)的红细胞变形性和膜稳定性。B组和A组的全血和红细胞变形性指数(DI)差异无统计学意义，而C组和D组的全血和RBC变形性指数(DI)随着苯肼治疗的抑制而降低。含氧化血红蛋白的RCG的DI值降低，膜的稳定性增强。这些发现表明，细胞骨架红细胞蛋白之间的相互作用将发生改变。The fr…在剪切力(750dyn/cm^2)和T50的作用下，当DI0.5(50%)时，A组为15.6±/-23秒，B组为20.25+/-2.63秒，C组为17.9+/-4.02秒，D2组为16.25+/-1.44秒。在-50μH2O连续负压下，研究了稀释的红细胞溶液通过镍网(孔径为4 mm)的过滤性能。与B、C、D组相比，B、C、D组红细胞的流速-压力曲线右移，红细胞的滤过性降低。与血影蛋白和肌动蛋白一起，红细胞骨架蛋白4.1在红细胞膜骨架结构的水平面上形成六方晶格复合体(网状网络)，通过对骨骼红细胞蛋白4.1的生化和结构研究，证实了钙调素/钙调素/钙离子的结合部位；4.4.细胞骨架红细胞蛋白的SDS-PAGE图谱无明显差异。5.Giemsa染色的红细胞涂片镜检发现，B组和D组有微小的差异。6.流式细胞仪检测膜联蛋白V阳性的红细胞百分率在D组显著增加，提示磷脂酰丝氨酸的出现，正常情况下局限于内脂质层，红细胞膜上糖蛋白(糖蛋白等)侧链上的坐骨酸引起的负电荷相互排斥，但唾液酸的减少和糖蛋白的缺陷导致红细胞膜上负电荷减少和聚集。用阴离子交换树脂和阳离子交换树脂测定各组红细胞的吸附能力，研究膜电荷的大小。4组间红细胞对阴阳离子交换树脂的吸附能力无明显差异。经Endo-β-半乳糖苷酶处理后，红细胞膜上的糖蛋白几乎完全去除，红细胞相互聚集。此外，激光衍射法观察到，红细胞表面糖蛋白特异性结合的Rectin处理降低了红细胞的变形性。结论：糖尿病透析患者红细胞的变形性和滤过性降低，红细胞表面糖蛋白的减少或缺陷可能参与了红细胞轮状的形成。糖尿病透析患者红细胞膜脂质b*层的不对称性与外层磷脂酰丝氨酸的增加有关。去除红细胞膜上的糖蛋白后，红细胞膜上的负电荷减少，红细胞容易相互聚集。暴露于氧化应激可降低红细胞的变形性，并改变红细胞与细胞骨架蛋白的相互作用。较少

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