机械张应变诱导的大鼠血管平滑肌细胞磷脂酰肌醇3激酶/蛋白激酶B信号通路相关基因的筛选  

IDENTIFICATION OF PI3K/Akt PATHWAY RELATED GENES IN VASCULAR SMOOTH MUSCLE CELLS SUBJECTED TO CYCLIC STRAIN

作　　者：姚庆苹[1] 严志强[1] 白玲[1] 姜宗来[1] 

机构地区：[1]上海交通大学力学生物学与医学工程研究所,上海200240

出　　处：《解剖学报》2007年第6期656-659,共4页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(10472071;10132020)

摘　　要：目的筛选机械张应变诱导的大鼠血管平滑肌细胞(VSMCs)磷脂酰肌醇3激酶/蛋白激酶B(PI3K/Akt)信号通路的相关基因。方法应用FX-4000T细胞应变加载系统,对大鼠主动脉VSMCs施加1Hz、10%张应变,Western blotting检测细胞在不同加载时间下Akt磷酸化水平的变化;用抑制消减杂交法(SSH),筛选给予PI3K的抑制剂Wortmannin和给予DMSO两组细胞在加载张应变24h后的差异表达基因片段,得到的消减杂交产物连接到T载体上,经过蓝白斑筛选,对所有阳性克隆进行菌液PCR筛选及测序,应用BLASTN进行序列比对。结果机械张应变改变了VSMCs磷酸化Akt水平;SSH所获得的54个克隆随机挑选30个送测序,得到10个不同差异表达序列标签(EST),发现6个EST代表的大鼠基因可能与机械张应变诱导的VSMCs PI3K/Akt信号通路相关,它们是:Highmobility group box1(HMGB1),Neural precursor cell expressed(Nedd4a),Cyclin-dependent kinase5(CDK5),Histonedeacetylase3(HDAC3),Ubiquitin-like1(Uble1a),Heterogeneous nuclear ribonucleoprotein H1(hnRNP)。结论SSH有效筛选到了机械张应变诱导的VSMCs PI3K/Akt信号通路的相关基因,为进一步研究机械张应变诱导的血管病理生理改变提供了资料。Objective To screen out the PI3K/Akt pathway related genes in vascular smooth muscle cells （VSMCs） subjected to cyclic strain. Method VSMCs of rat aorta were subjected to cyclic strain （ 10 %, 1 Hz） by using a FX-4000T system. Phosphorylation of Akt was examined by Western blotting. Suppression subtractive hybridization （SSH） method was employed to analyze the differently expressed cDNA sequence between the strained VSMCs pretreated with and without wortmannin, a specific inhibitor of PI3K. These fragments were ligated with T vectors, screened through the blue-white screening system to establish cDNA library, and was sequenced and analyzed in GenBank with BLASTN search. Result Level of Akt phosphorylation of VSMCs was significantly enhanced by the mechanical strain compared with the control. Ten different expressed sequence tags （EST） were gained after sequencing 30 clones randomly selected from 54 white clones. There may be six genes related with the mechanical strain and cell signal PI3K/Akt, such as High mobility group box 1 （ HMGB1 ）, Neural precursor cells expressed （Nedd4a）, Cyclin-dependent kinase 5 （CDKS）, Histone deacetylase 3 （ HDAC3）, Ubiquitin-like 1 （Ublel a） and Heterogeneous nuclear ribonucleoprotein H1 （hnRNP）. Conclusion SSH is an effective and reliable approach to screen out the genes related with the mechanical strain and PI3K/Akt pathway in VSMCs.

关 键 词：机械张应变 血管平滑肌细胞 抑制消减杂交法 磷脂酰肌醇3激酶 蛋白激酶B信号通路 大鼠 

分 类 号：Q952[生物学—动物学]