Role of Eosinophil in the Onset and Chronicity of Ulcerative Colitis

嗜酸性粒细胞在溃疡性结肠炎的发病和慢性中的作用

• 批准号: 08457169
• 负责人: MAKIYAMA Kazuya
• 金额: $ 2.37万
• 依托单位: Nagasaki University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457169/
• 关键词: ulcerative colitis; eosinophil cationic protein : ECP; purification of ECP from normal human eosinophils; contractile effect of ECP to intestinal amooth muscle; contribution to chronic inflammation; 平滑筋収縮機構; Eosimophil cationic protein; 分離精製; 平滑筋収

Background : In ulcerative colitis, the colonic mucosa exhibited the marked infiltration of activated eoslnophils with the secretoly form of eosinophil cationic protein(ECP), one of the eosinophll granule proteins. Although it Is strongly suggested that ECP is closely associated with the inflammatory process of ulcerative colitis, the direct effect of ECP to the intestine remains obscure.To elucidate the mucosal damage caused by ECP, purification of ECP from normal human eosinophils was performed, and then, to examine the effect of ECP on the contraction of the smooth muscle of the glnea pig small intestine.Methods : 1. Purification of ECP : ECF was purified from the granules of human buffy coat eosinophils obtained from healthy Individuals. Method of Peterson et al. (1988) was followed for purification The procedure included 1) pretreatment : (1) separation of leukocytes by Dextran treatment from approximately 2l buffy coats of normal subjects, (2) separation of granules by homogenizi … More ng of leukocytes, and (3) 20-fold concentration of the granules by ultra-filtration, and 2) cabin chromatography : (1) gel filtration on Superdex 75, (2) ion exchange chromatography on SOURCE.and (3) chelating chromatography on zinc-chelating Sepharose. ECP was measured by RIA (Pharmacla ECP RIA kit). 2. Examinatlon of the contraction : The strips of the guinea pig small intestine (2cm long were removed from a region 10cm proximal from the lleo-cecal Junction) were placed in a oragan bath In the presence of Krebs-Ringer solution. ApproxImately 600 mg of resting tension was applied and was kept constant. ECP1O mug/l was added to the oragan bath, and mechanical activity was recorded by means of an isometric transducer. In addition, examination whether atropine inhibits the contraction effect of ECP was performed.Results : 1. Purification of ECP : The ECP activity at the final stage of separating process showed a definite I)COk at fractions No. 47(the concentration of ECP was 4292 mug/l) and No. 48 (that was 3805 mug/l) ECP was existent in the area of molecular weight l6kD to 21.4kD in electrophoresis, and it was considered that a high degree of ECP purification was available. 2. Examination of the contraction : After addition of 10 mug/l ECP, which was the cut off concentration of normal serum, to the organ bath, the transient contraction was observed. The width of the contraction Inducted by ECP was approximately 13% of that of inducted by 10^-^6 M acetylcholine. Furthermore, the ECP contraction effect to the smooth muscle of guinea-pig were inhibited partially by atropine at preliminary experiment.Conclusion : This is the first demonstration of the effect of ECP to the intestine. ECP conrtacts the smooth of the guipan pig intestene and may contribute the pathopbysiology of ulcerative colitis. Less

背景资料：在溃疡性结肠炎中，结肠粘膜表现出明显的嗜酸性粒细胞浸润，嗜酸性粒细胞阳离子蛋白（ECP）的分泌形式，嗜酸性粒细胞颗粒蛋白之一。虽然ECP与溃疡性结肠炎的炎症过程密切相关，但其对肠道的直接作用尚不清楚，为了阐明ECP对肠粘膜的损伤作用，本实验从正常人嗜酸性粒细胞中分离纯化ECP，并观察ECP对猪小肠平滑肌收缩的影响。ECP的纯化：从健康个体获得的人血沉棕黄层嗜酸性粒细胞的颗粒中纯化ECF。采用Peterson等（1988）的方法进行纯化。方法包括：1）预处理：（1）用右旋糖酐处理从正常受试者约2L血沉棕黄层中分离白细胞，（2）用均质化法分离颗粒，（3）用葡聚糖处理从正常受试者约2L血沉棕黄层中分离颗粒。 ...更多信息 ng白细胞，和（3）通过超滤将颗粒浓缩20倍，和2）舱式色谱法：（1）在Superdex 75上的凝胶过滤，（2）在SOURCE上的离子交换色谱法和（3）在锌螯合琼脂糖上的螯合色谱法。采用放射免疫法（Pharmacla ECP RIA Kit）测定ECP。2.收缩的检查：将豚鼠小肠条（从回盲肠连接处近端10 cm处取出2cm长）置于含Krebs-Ringer溶液的oragan浴中。施加约600 mg静息张力并保持恒定。将ECP 10 μ g/l加入到oragan浴中，并通过等距传感器记录机械活动。此外，还观察了阿托品是否抑制ECP的收缩作用。ECP的纯化：在分离过程的最后阶段，ECP活性显示出一定的I）在47号组分（ECP浓度为4292 μ g/l）和48号组分（ECP浓度为3805 μ g/l）处的COk。电泳中ECP存在于分子量为16 kD至21.4kD的区域，认为ECP的纯化程度较高。2.收缩的检查：向器官浴中加入10 μ g/l ECP（正常血清的截止浓度）后，观察到短暂收缩。ECP引起的收缩幅度约为10^-^6 M乙酰胆碱引起的收缩幅度的13%。初步实验表明，阿托品可部分抑制ECP对豚鼠离体肠平滑肌的收缩作用。ECP使贵潘猪小肠平滑肌收缩，可能参与溃疡性结肠炎的病理生理过程。少

项目成果

牧山 和也: "厚生省特定疾患難治性炎症性腸管障害調査研究班 平成9年度研究報告書" 厚生省特定疾患難治性炎症性腸管障害調査研究班 班長 下山孝, 230 (1998)

Kazuya Makiyama：“保健福祉部特定疾病顽固性炎症性肠疾病研究小组 1997 年度研究报告” Takashi Shimoyama，保健福祉部特定疾病顽固性炎症性肠疾病研究小组组长，230 (1998)

Makiyama K.: Annual Report of the Research Committee of Inflammatory Bowel Disease The Ministry of Health and Welfare of Japan. 1997's Report. Purification of eosinophil cationic protein (ECP) and its significance, (1998)

Makiyama K.：日本卫生福利部炎症性肠病研究委员会的年度报告。