缺氧后处理诱导大鼠心肌细胞蛋白质差异表达  

Proteomic Differential Expression of Rat Cardiomyocytes Induced by Hypoxic Postconditioning

作　　者：徐菲菲[1] 孙胜[1] 刘秀华[1] 

机构地区：[1]中国人民解放军总医院病理生理研究室,北京100853

出　　处：《中国微循环》2009年第2期77-84,共8页Journal of Chinese Microcirculation

基　　金：国家自然科学基金(No.30670822);国家重点基础研究发展计划(No.2007CB512003);解放军总医院苗圃基金(07MP68)

摘　　要：目的研究缺氧后处理（hypoxic postconditioning，H-postC）与缺氧／复氧（hypoxia／reoxygenation，H／R）诱导的大鼠心肌细胞蛋白质差异表达，旨在从内源性蛋白表达变化的角度阐明后处理保护的分子机制。方法原代培养Sprague-Dawley（SD）乳鼠心肌细胞随机分为3组（均n=4）：（1）缺氧／复氧（H／R）组：将细胞置于缺氧仓内，通入95％N2-5％CO2混合气2h后，37℃常氧孵育24h；（2）缺氧后处理（H-postC）组：细胞置于缺氧仓内2h，复氧10mitt／缺氧20rain，反复2次，37℃常氧孵育24h；（3）对照组（control）：细胞置于CO2孵箱37℃常氧孵育至实验结束。提取各组心肌细胞蛋白质，经双向电泳分离、考马斯亮蓝染色后扫描分析蛋白质表达变化，选取9个差异显著的蛋白点进行胶内酶切、肽质量指纹图谱分析和NCBInr数据库检索。结果双向电泳可分离（553±31）个蛋白质，点匹配率为80．1％±1．5％。与对照组相比，6种蛋白质在H-postC组表达升高，而在H／R组表达降低；1种蛋白质在H-postC组和H／R组表达均降低，而H-postC组降低更为明显；1种蛋白质在H-postC组和H／R组表达均升高，而H-postC组升高更为明显；1种蛋白质仅在H-postC组表达升高。质谱鉴定的9种蛋白质可分为5类：①保护性伴侣分子：DJ-1蛋白、αB-晶体蛋白、热休克蛋白27；②细胞凋亡调节分子：星型胶质细胞磷蛋白15。非受体6型蛋白酪氨酸磷酸酶；③核糖核蛋白复合体组分：不均质核内核糖核蛋白K；④线粒体能量代谓十相关分子：线粒体ATP合酶D链、细胞色素C氧化酶亚单位VIa；⑤细胞骨架蛋白：β-肌动蛋白。结论H-postC可能通过上调保护性分子伴侣、调节细胞凋亡相关的信号分子、稳定核糖核蛋白复合体结构、维持线粒体功能活性、稳定细胞骨架而发挥心肌保护作用。Objective To explore proteomic changes in rat myocardial ceils induced by hypoxic postconditioning（H-postC） and hypoxia/reoxygenation（H/R）. Methods Cultured myocardial cells of neonatal Sprague-Dawley rats were randomly divided into three groups（ n = 4） ： （1） H/R group： hypoxia （95% N2 -5% CO2） for 2 h followed by reoxygenation（95 % air - 5 % CO2） for 24 h. （2） H-postC group： 2 cycles of reoxygenation for 10 min/hypoxia for 20 min at the beginning of reoxygenation after 2 hours＇ hypoxia. （3） Control group. After protein extraction, 2-D electrophoresis（2-DE） was employed to separate proteins, and 9 protein spots expressed differentially were picked out and subjected to tryptic in-gel digest and matrix-assisted laser desorption/ionization-time of/light mass spectrometry（MALDI-TOF） for identification. Results More than 553 protein spots were detected on each gel and a match rate 80.1% ± 1.5% was achieved. Compared with control, 6 proteins increased after H-postC, while decreased after H/R. 1 protein decreased after both H-postC and H/R, but the descent degree was more obviously after H-postC. 1 protein increased after both H-postC and H/ R, but the height degree was more obviously after H-postC. 1 protein increased only after H-postC. After mass spectrometry identification, these 9 proteins were classified into 5 species as follows： ①protective chaperones： heat shock protein27（HSP27）, alpha B-crystallin and DJ-1 protein; ②apoptosis regulatory molecules： phosphoprotein enriched in astrocytes 15（predicted） isoform 2 and protein tyrosine phosphatase, non-receptor type 6; ③compositions of ribonucleoprotein complex： Heterogeneous nuclear ribonucleoprotein K, isoform b; ④mitochondrial energy metabolism relative molecules： ATP synthase D chain, mitochondrial and cytochrome c oxidase subunit VIa（AAl-118）; ⑤cytoskeleton protein： β-actin. Conclusion H-postC might induce protective chaperones upregulation, adjust apoptosis regulatory molecules

关 键 词：缺氧后处理 心肌细胞 蛋白质组 双向电泳 质谱 

分 类 号：Q24[生物学—细胞生物学]