细胞肥大过程中hhLIM的亚细胞定位变化及其对细胞骨架的影响  

Subcellular Localization of hhlim Gene product and Its Effect on Cytoskeleton in Cardiac Hypertrophy

作　　者：郑斌[1] 温进坤[1] 韩梅[1] 

机构地区：[1]河北医科大学基础医学研究所,河北省医学生物技术重点实验室,石家庄050017

出　　处：《中国生物化学与分子生物学报》2005年第2期255-261,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家重点基础研究发展规划项目 (973 )基金 (No .G2 0 0 0 0 5 690 5 ) ;国家自然科学基金 (No .3 0 3 0 0 13 2 );河北省自然科学基金 (No .2 0 0 40 0 0 64 4)资助~~

摘　　要：hhlim (humanheartlim)是从人胎心cDNA文库中筛选克隆的一个新基因 ,作为LIM家族的新成员参与心肌肥大的发生发展过程 .为了进一步研究hhLIM在心肌肥大发生过程中的作用 ,以C2C12细胞为研究对象 ,以心肌肥大强效刺激因子内皮素 1(ET 1)为诱导因素 ,探讨hhLIM与肌动蛋白的相互作用及其影响细胞骨架的分子机制 .RT PCR、Western印迹和细胞免疫荧光分析结果表明 ,心肌肥大刺激因子ET 1在诱导心肌肥大标志基因BNP和肌动蛋白表达的同时 ,使hhLIM蛋白在C2C12细胞胞核与胞质之间进行重新定位 .激光共聚焦显微镜观察结果显示 ,hhLIM与肌动蛋白在胞质中共定位 .蛋白分步提取、鉴定及hhLIM与F肌动蛋白结合与沉降实验证明 ,hhLIM多存在于细胞骨架及其相关蛋白部分 ,在体外可与F肌动蛋白共结合 .这些结果表明 ,胞质中的hhLIM作为细胞骨架相关蛋白与肌动蛋白相互作用 .进一步研究hhLIM与细胞骨架的关系时发现 ,hhLIM过表达可使C2C12细胞的骨架变成致密网状纤维并使其对细胞松弛素导致的细胞骨架解聚产生一定的抵抗作用 ,抑制hhLIM表达则使细胞骨架稀疏 ,结构模糊 .提示hhLIM参与细胞骨架组织及重构的机制与其结合并稳定F肌动蛋白有关 .hhlim is a new heart-related gene cloned from embryonic heart cDNA libraries whose product plays an important role in cardiac hypertrophy. In order to further explore the role of hhLIM in cardiac hypertrophy, C2C12 cells were treated with hypertrophic agonist endothelin-1 to induce cell hypertrophy, and then the interaction between hhLIM and actin and its effect on cytoskeleton were studied. RT-PCR, Western blotting and immunofluorescence assays identified that endothelin-1 could induce the expression of cardiac hypertrophy marker gene BNP and α-actin, and enable hhLIM protein redistribute between nucleus and cytoplasm. Confocal microscopy images showed that hhLIM protein in the cytoplasm co-localized with actin. Protein extracts from different cellular compartments and their co-sedimentation with F-actin demonstrated that hhLIM existed in the extracts of cytoskeleton-associated protein and co-sedimentated together with F-actin in vitro. This is consistent with immunofluorescence assay indicating hhLIM protein acting as a cytoskeleton-associated protein. Further study of relationship between hhLIM and cytoskeleton suggested that overexpression of hhLIM made the cytoskeleton highly organize and prevented the cytoskeleton from depolyperization by cytochalasin B. Inhibition of hhLIM expression by antisense transcripts induced the cytoskeleton loose and accelerated actin depolyperization. In conclusion, the molecular mechanism of hhLIM-participated in the cytoskeleton organization and rearrangement is related to its association with and stabilization of actin cytoskeleton.

关 键 词：hhlim基因 C2C12细胞 肌动蛋白 细胞骨架 

分 类 号：Q78[生物学—分子生物学] Q245