iNOS在不同种属血管细胞中诱导表达差异的比较研究

为了探讨不同种属血管细胞中ｉＮＯＳ基因诱导表达的差异及其分子机制，采用Ｎｏｒｔｈ－ｅｒｎ印迹、电泳迁移率改变分析（ＥＭＳＡ）和细胞转染实验对ｉＮＯＳ基因在人、牛、大鼠血管内皮细胞（ＥＣ）和兔、大鼠平滑肌细胞（ＶＳＭＣ）中的诱导表达和转录调控机制进行了研究．结果显示，ＩＬ－１β可诱导人、大鼠的ＥＣ和大鼠的ＶＳＭＣ表达ｉＮＯＳ，但对兔ＶＳＭＣ和牛ＥＣ无诱导作用．在ＩＬ－１β＋ＴＮＦ－α＋ＬＰＳ作用下，人和大鼠血管细胞ｉＮＯＳ的表达活性显著高于牛和兔，同一种属动物的ＶＳＭＣ比ＥＣ更易被诱导活化．用含有大鼠ｉＮＯＳ基因上游－１０３７～－４３８片段的报告基因转染这些细胞，经细胞因子和ＬＰＳ处理后，被转染细胞的ＣＡＴ活性变化与细胞的ｉＮＯＳ表达活性相一致．上述结果表明，ｉＮＯＳ表达调节具有种属特异性和细胞特异性．ＥＭＳＡ证实在不同种属的ＥＣ和ＶＳＭＣ中，与ｉＮＯＳ基因表达调控区（－１０３７～－７８７）相互作用的蛋白因子是不均一的．提示不同种属血管细胞内特异转录因子的种类及浓度不同和（或）反式因子与顺式元件相互作用的方式不同可能是这种差异的分子基础．

Comparative Study of iNOS Gene Expression in Vascular Cells of Different Species

To determine the expression difference of iNOS gene in the different species and cells,iNOS gene expressions and their regulatory mechanisms in rat,human,bovine and rabbit vascular endothelial cells(EC)and/or smooth muscle cells(VSMC)were studied by Northern blotting,chloramphenicol acetyltransferase (CAT) assay and electrophoretic mobility shift assay (EMSA).The iNOS gene was induced in human EC,rat EC and rat VSMC,but not detected in bovine EC and rabbit VSMC following IL 1β stimulation.The combination of IL 1β+TNF α+LPS together induced higher levels of iNOS expression in human and rat vascular cells of human and rat than those of bovine and rabbit ones.VSMC iNOS in response to cytokines and LPS showed greater activity compared to EC iNOS in the same species.After the report plasmids with -1073～-438 fragment of rat iNOS gene 5′ flanking region were transfected into the above cells,CAT activities detected in the cells were accordant with the levels of iNOS expression under the equal stimulation.EMSA with rat iNOS gene -1037～-787 region and nuclear extracts from EC and VSMC showed that the electrophoretic patterns were different.These results suggested that cell and specy specific mechanisms existed in the control of iNOS expression.The molecular mechanisms of those differences might be attributable to the heterogenecity of nuclear factors and /or the different interaction of trans acting factors with cis acting elements.