Chaqour B, Howard P S, Macarak E J
University of Pennsylvania, Department of Anatomy Histology, Philadelphia 19104, USA.
Mol Cell Biochem. 1999 Jul;197(1-2):87-96. doi: 10.1023/a:1006966530553.
Physical forces induce profound changes in cell phenotype, shape and behavior. These changes can occur in vascular structures as a result of pressure overload and their effects can be seen in atherosclerotic vessels in which smooth muscle cells have undergone hyperplastic and hypertrophic changes. At the molecular level, mechanical stimuli are converted into chemical ones and lead to modulation of gene expression and/or the activation of a new repertoire of genes whose encoded proteins help the cells to adapt to their microenvironment. In this study, we have used a two primer-based mRNA differential display technique to identify candidate mechano-responsive genes in pulmonary artery smooth muscle cells. As compared to the original method described by Liang and Pardee, this technique uses two arbitrary primers instead of an anchored oligo(dt) plus an arbitrary primer in the polymerase chain reaction. The chief advantages of these modifications are an increase in the efficiency of the amplification and in the identification of differentially expressed clones. Using this approach, we compared the pattern of expressed genes in cells cultured under static conditions with those in cells that were mechanically stretched (1 Hz) for 24 h in a well-defined in vitro mechanical system. Three candidate genes that showed reproducible differences were chosen for further characterization and cloning. One clone was under expressed in stretched cells and had a DNA sequence with 90% homology to the human fibronectin gene. Two other clones were highly expressed in stretched cells and had a 92% and a 83% sequence homology with human platelet-activating factor (PAF) receptor and rat insulin-like growth factor-I (IGF-I) genes respectively. Northern blot analysis confirmed low levels of fibronectin mRNA transcripts in stretched cells. In contrast, accumulation of PAF receptor mRNA occurred 30 min after mechanical stretch was initiated whereas IGF-I mRNA levels peaked at 8 h. Both mRNA levels were sustained for up to 24 h of mechanical stretching. These results demonstrate the usefulness of the two primer-based mRNA differential display that enabled us to identify and characterize alterations at the level of gene expression among matrix proteins, G-protein coupled receptors and growth factors, each of whose response to mechanical strain is different. A more complete understanding of these responses will provide further insight into the pathologic processes associated with hypertension and atherosclerosis.
物理力可引起细胞表型、形状和行为的深刻变化。由于压力过载，这些变化可发生在血管结构中，其影响可见于动脉粥样硬化血管，其中平滑肌细胞发生了增生性和肥大性变化。在分子水平上，机械刺激被转化为化学刺激，并导致基因表达的调节和/或一组新基因的激活，其编码的蛋白质有助于细胞适应其微环境。在本研究中，我们使用了基于两种引物的mRNA差异显示技术来鉴定肺动脉平滑肌细胞中候选的机械反应基因。与Liang和Pardee描述的原始方法相比，该技术在聚合酶链反应中使用两种任意引物，而不是一个锚定的oligo(dt)加上一个任意引物。这些修改的主要优点是提高了扩增效率和差异表达克隆鉴定效率。使用这种方法，我们比较了在静态条件下培养的细胞与在明确的体外机械系统中以1 Hz频率机械拉伸24小时的细胞中表达基因的模式。选择了三个显示出可重复差异的候选基因进行进一步表征和克隆。一个克隆在拉伸细胞中表达下调，其DNA序列与人纤连蛋白基因有90%的同源性。另外两个克隆在拉伸细胞中高表达，分别与人血小板活化因子(PAF)受体基因和大鼠胰岛素样生长因子-I (IGF-I)基因有92%和83%的序列同源性。Northern印迹分析证实拉伸细胞中纤连蛋白mRNA转录本水平较低。相反，机械拉伸开始30分钟后PAF受体mRNA开始积累，而IGF-I mRNA水平在8小时达到峰值。两种mRNA水平在机械拉伸长达24小时内持续存在。这些结果证明了基于两种引物的mRNA差异显示的有用性，使我们能够鉴定和表征基质蛋白、G蛋白偶联受体和生长因子之间基因表达水平的变化，它们对机械应变的反应各不相同。对这些反应的更全面理解将为与高血压和动脉粥样硬化相关的病理过程提供进一步的见解。
