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New Method for Detection Ischemic Heart Cell Damage

检测缺血性心脏细胞损伤的新方法

基本信息

批准号：
63870037
负责人：
YASUDA Hisakazu
金额：
$ 5.95万
依托单位：
Hokkaido University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B).
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63870037/
关键词：
Fatty acid binding protein Free fatty acid Hypoxic cell injury Myocardial infraction Cardoprotestive drugs FABP 心筋肥大心筋虚血ラジオイレムアッセイ

项目摘要

Fatty acid binding protein (FABP) is thought to play an important role as a carrier protein in intracellular transportation of fatty acids and lipid metabolism. It accounts for several percentage of soluble protein. Because the molecular weight of FABP is low (mol wt 14000), it may be released from the cells in case of the hypoxic myocyte as a maker of the hypoxic cell injury. New born rat myocytes were incubated under hypoxic treatment for 6 hrs, then the release of FABP and CPK were measured. Delipidation of samples and assay of fatty acid binding were carried out using a Lipidex 1000, according to the method of Glatz et al.. Lipidex 1000, a 10% (w/w) substituted hydroxyalkoxypropyl derivative of Sephadex G-25, removes unbound fatty acids from protein-fatty acid complexes at 0^ﾟC, and removes all fatty acids at 37^ﾟC from aqueous solutions according to protein-lipid interaction kinetics. For delipidation, samples (30mg) were subjected to chromatography on the Lipidex column (1.5 x 7 … More cm) equilibrated with 10mM sodium phosphate buffer (pH 7.4) at 37^ﾟC. The column was eluted with the same buffer and all the proteins were recovered in the void bolume. For the assay of fatty acid binding, samples were incubated with various concentrations of 14C-labeled fatty acids (Amersham ; Arlington Heights, IL) in a polyethylene tube in 10 mM sodium phosphate buffer (pH 7.4 ; final volume 0.45ml) for 10 min at 37 C. Then, to remove unbound fatty acids, the tubes were cooled on ice and ice-cold Lipidex/buffer suspension (1 : 1 v/v ; 0.05ml) was added and incubated for another 10min at 0^ﾟC. Fatty acid binding was calculated from the amount of radioactivity present in the supernatant after centrifugation of the tubes, and was expressed as pmol/ug of protein.The cell death ratio during hypoxygenation increased since 4 h and rose to 80% at 6 h, but it was only 8% under aerobic conditions. FABP was detected at 1 h, rapidly increased and reached plateau at 4 h. On the other hand CPK release was negligible during 6h. Ca-antagonist and blocker inhibited the release of FABP and prevented the cell death. These result shows FABP is of use as a maker of myocardial cell injury, and hypoxic cell injury is mediated through adrenergic receptor. Less

脂肪酸结合蛋白（FABP）作为一种载体蛋白，在脂肪酸的细胞内转运和脂质代谢中起着重要作用。它占可溶性蛋白质的几个百分比。由于FABP的分子量较低（mol wt 14000），在缺氧心肌细胞的情况下，其可以从细胞中释放，作为缺氧细胞损伤的标志物。新生大鼠心肌细胞缺氧培养6小时，测定FABP和CPK的释放。根据Glatz等人的方法，使用Lipidex 1000进行样品脱脂和脂肪酸结合测定。Lipidex 1000是Sephadex G-25的10%（w/w）取代羟基烷氧丙基衍生物，可在0 ° C下从蛋白质-脂肪酸复合物中去除未结合的脂肪酸，并根据蛋白质-脂质相互作用动力学在37 ° C下从水溶液中去除所有脂肪酸。对于脱脂，将样品（30 mg）在Lipidex柱（1.5 x 7）上进行色谱分析 ...更多信息 cm），在37 ° C下用10 mM磷酸钠缓冲液（pH 7.4）平衡。用相同的缓冲液洗脱柱，并将所有蛋白质回收到空柱中。对于脂肪酸结合的测定，将样品与不同浓度的14 C-标记的脂肪酸（阿默舍姆;阿灵顿Heights，IL）在聚乙烯管中的10 mM磷酸钠缓冲液（pH7.4;终体积0.45 ml）中于37 ℃温育10分钟。然后，为了除去未结合的脂肪酸，将管在冰上冷却，加入冰冷的Lipidex/缓冲液悬浮液（1：1 v/v ; 0.05ml），并在0 ° C下再孵育10分钟。脂肪酸结合率由离心管后上清液中的放射性量计算，并表示为pmol/ug蛋白质。缺氧期间的细胞死亡率从4 h开始增加，6 h时上升至80%，但在有氧条件下仅为8%。FABP在1h检测到，迅速升高，4 h达平台期。另一方面，CPK释放在6小时内可忽略不计。Ca ~（2+）拮抗剂和阻断剂可抑制FABP的释放，防止细胞死亡。提示FABP是心肌细胞损伤的标志物，缺氧性心肌细胞损伤是通过肾上腺素能受体介导的。少