用差异显示PCR法筛选与血管外膜细胞表型转化相关的基因

Identification of genes related to cell phenotypic transition by differential display analysis

为筛选血管外膜成纤维细胞 (adventitialfibroblast,AF)与肌成纤维细胞 (myofibroblast,MF)间表型转化有关的基因 ,实验建立了大鼠胸主动脉AF和MF两种细胞模型 ,用差异显示聚合酶链反应 (DD PCR)技术获得表达差异片段 ,对差异片段进行克隆和测序分析 ,并用定量PCR和Northernblot对差别显示结果进行验证。用反义核酸转染技术观察骨桥蛋白 (osteopontin ,OPN)对AF迁移的影响。结果表明 ,两种表型细胞存在明显的基因表达差异 ,其中一个在MF下调的差异片段与GenBank中NADH脱氢酶亚单位 5 (NADHdehydrogenasesubunit 5 ,Nd5 )基因高度同源。另一个在MF上调的差异片段与OPN基因同源。上述差异表达结果被定量PCR及Northernblot证实。此外还有 4个表达序列标志 (expressedsequence tag,EST)在GenBank中未查到同源序列。反义OPN寡脱氧核苷酸可抑制AF的迁移活动。结果提示 ,AF转化为MF可能与ND5基因下调、OPN上调及其它未知基因的表达改变有关。应用反义技术适度抑制OPN表达在防治血管重塑中具有重要作用。

To identify the genes that are differentially expressed during the phenotypic transition from vascular adventitial fibroblasts to myofibroblasts, the adventitial fibroblasts were cultured from rat thoracic aorta, and myofibroblasts were obtained by treatment of fibroblasts with TGF-β1. Differential display PCR (DD-PCR) was used to screen for differentially expressed genes by comparison of mRNA extracted from the two cell populations. Bands upregulated or downregulated on DD gels were excised, reamplified, cloned and sequenced. DD results were verified by quantitative PCR and Northern blot analysis.Antisense oligonucleotide was transfected to study the effect of osteopontin on migration of AF. Differential display showed a significant difference in gene expression profile between the two cell types. A transcript that was downregulated in myofibroblasts showed high DNA sequence homology to part of the gene for NADH dehydrogenase subunit 5. An upregulated transcript showed significant sequence homology to osteopontin gene. Quantitative PCR and Northern blot analysis confirmed the DD results. Among the other differential bands detected, 4 candidate sequences showed no homology to the known genes. The AF numbers of migration were significantly decreased by use of OPN antisense oligonucleotide. This study suggests that the downregulation of gene encoding NADH dehydrogenase subunit 5 and upregulation of osteopontin gene and several other unknown genes may be involved in the phenotypic transition of adventitial fibroblasts to myofibroblats. Inhibition of the expression of OPN may play an important role in the process of vascular remodeling.