化学性缺氧对大鼠心肌细胞葡萄糖转运蛋白4转位的影响.pdf

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化学性缺氧对大鼠心肌细胞葡萄糖转运蛋白4转位的影响
罗佳,于波,关付
【摘要】目的研究化学性缺氧対大鼠心脱细胞萄糖转运蛋白4(Clu4)转位的影响。方法原代培养的大
鼠心室肌细胞被随机分为对照组、胰岛素组、叠氩钠(特异性细胞色素C氧化酶抑制剂)模拟的化学性缺氧组,然后
利用差速离心及蔗糖密度梯度分离出细胞膜和细胞内膜,再用 Westen-Blot法分别测定膜组分中Clut4的表达量。结
果同对照组相比,胰岛素组、化学性缺氧组大鼠心肌细胞膜上Clu4表达量均显著增加,差别有显著性意义(P<
0.05), Western-Blot法測定平均灰度值:对照组为(26.8±2.3),胰岛素组为(61.1±3.8),化学性缺氧组为
(47.2±1.5);而细胞内膜Clut4含量相应下降(P<0.05),提示心肌细胞发生了Clu4转位。结论胰岛素和缺氧可
诱导原代培养的大鼠心肌细胞Glut4发生不同程度的转位。
【关键讽}心肌细胞;大鼠;化学性缺氧;Clut4转位
【中图分类号】R542.2【文献标识码A【文章编号】1007-9572(2007)11-0897-03
Effect of Chemical Hypoxia on Trans ation of Glucose Transporter-4 in Rat Cardiomyocytes LUO Jia, YU Bo, GUAN
Fu. Shenzhou Hospital Afiliated to Shenyang Medical College, Shenyang 110002, China
Abstract Objective To investigate the effect of chemical hypoxia on the translocation of glucose transporter-4
(Glut 4 )in rat cardiomyo tes Methods Pimay culured Wistar rat myocar es were randomly divided ascontrol group
insulin group cemical hypoxia group induced by sodium azide (Nan3) a specific inhibior of cytochrome C oxidase. Then
differential centrifugation and discontinuous sucrose gradient were taken to separate plasma membranes(PM) and intracellular
membranes(IM and the expressions of Glut4 in these separated membrane fractions were determined with Western blot, Re
sults Compared with the control group, the expressions of Glut4 in the PM in insulin group and chemical hypoxia group were
obviously increased, with a significant difference(P <0.05), and the average gray values detected by Western blot were
(6.8+.)in the cool, (61 13.8 in the insulin, (47.2*1.5)in the hypoxia while the expressions of Glut4 in the
IM were decreased (P < O. 05), indicating aocation of Gl4 rom an intracellular compartment to the plasma membrane
Conclusion Both insulin and Nan3-induced chemical hypoxia can cause translocation of Glut4 molecules to a different degrees
In the primary cultured rat cardiomyocyte
I Key word) Cardiomyocyte; Rat; Chemical hypoxia; Glut4 translocation
心肌缺血/缺氧时葡萄糖的摄取和利用会显著增加り。各后剪成约1mm的碎块。用0.1%胰蛋白酶溶液37℃反复消
种临床和实验结果表明,急性缺血/缺氧期间葡萄糖摄取和代化组织碎块,向消化所得细胞沉淀中加入含10%胎牛血清的
谢增加对维持心肌舒缩功能、降低心肌酶释放、促进再灌注损DMEM培养液制成细胞悬液,然后以10°个细胞/ml的密度接
伤的恢复有重要作用?。心肌葡萄糖的摄取和转运主要与细种于培养瓶中,置37℃,10%C02培养箱中静止培养,并利
胞膜上易化性葡萄糖转运蛋白(Guts)数量有关,心肌最主用差速黏附技术提纯心肌细胞。根据培养液pH改变情况,每
要的Cus是Clu4。研究证明胰岛素、缺血、收缩“等隔2~3d更换培养液1次。培养1周左右,细胞单层融合,
可通过激发Glu4由细胞内膜向细胞膜转位,进而易化葡萄糖搏动同步化,形成功能性合体细胞。
转运。本文以体外原代培养的大鼠心肌细胞为研究对象,观察1.2制备缺氧模型及施加刺激因素原代培养大鼠心肌细胞
叠氮钠模拟的化学性缺氧对心肌细胞Cut4转位的影响,冒在形成单层同步搏动后,弃除原培养液,随机分为3组。分别施
从细胞水平探讨缺氧心肌葡萄糖转运机制。
加以下试验因素:对照组加DMEM培养液(不含胎牛血清
材料与方法
37℃孵育1h;胰岛素组加20mM胰岛素37℃孵育30min;
11大風心肌细胞原代培养取出生两天的雄性 Wistar大風叠氮钠模拟的化学性缺氧组加1mM叠氮钠37℃解育3h。实
20只,引颈处死。无菌条件下取出心脏,留取心室肌,冲洗
验中胰岛素和叠氮钠均用与对照组等量的不含胎牛血清的
基金项目:国家自然科学甚金资助项目(3044054);辽宁省教育
DMEM培养液溶解。
庁高等学校科学研究项目(202013149
1.3细胞膜与细胞内膜的分离孵育结束后利用胰酶消化法
作者单位:110002迁宁省沈阳市,中国医科大学第一临床学院循
重新将附壁细胞制成悬液,离心收集沉淀的细胞。将细胞置于
环内科
裂解液(inmM:250蔗糖,20 HEPES,PH7.4,2ECTA
NaN,和新鲜加人的蛋白酶抑制剂)中,于4℃超声破碎细胞,