摘要
我们以往的研究表明,TGF-β1可以诱导血管外膜成纤维细胞(adventitiai fibroblasts,AFs)向肌成纤维细胞(myo- fibroblasts,MFs)分化。为寻找可能涉及MF分化的基因,本实验采用寡核苷酸芯片技术动态检测细胞表型转化过程中基因表达的变化,实时定量RT-PCR验证芯片结果。在芯片上的15866条总探针组中,2121个探针组在TGF-β1刺激后至少一个时间点的表达发生2倍以上变化,其中1318个基因表达上调,761个基因表达下调,还有少数基因(42个)在不同的时间点既有上调又有下调表达。在1231个已知功能基因中,分泌磷蛋白1(secreted phosphoprotein 1,APPI)、Rho- associated coiled-coil forming kinase 2(ROCK2)的表达趋势与标志基因α-平滑肌肌动蛋白(α-SM-actin)的表达趋势相同,TGF-β1诱导MF分化过程中上调了电压门控性钾通道Shal家族成员2(potassium voltage-gated channel,Shal-related family and mem- ber 2,KCND2)的表达,这些基因参与了MF的分化；此外,还发现内皮素1(endothelin 1,EDN1)、补体成分、NADPH氧化酶4(NADPH oxidase 4,NOX4)和NAD(P)H dehydrogenase,quinone 1(NQO1)可能参与了MF分化。本实验用寡核苷酸芯片技术验证了通过其它技术证实的同MF分化相关的基因,并发现了新的涉及该过程的基因,基因表达谱研究有利于鉴定参与细胞分化的基因和通路。
Our previous study demonstrated that TGF-β1 could induce the differentiation of vascular adventitial fibroblasts （AFs） to myofibroblasts （MFs）. The aim of this study was to identify the genes which might be responsible for the cell phenotypic change using genechips. Cultured rat AFs were treated with TGF-β1 （10 ng/ml） for 0 min, 5min, 15min, 2 h, 12 h and 24 h, respectively. Then the cells were gathered to prepare total RNA. We examined TGF-βl-induced gene expression profiling using Affymetrix oligonucleotide microarrays and analyzed data by GCOS 1.2 software. Moreover, expressional similarity was measured by hierarchical clustering. Some of genechip results were confirmed by real-time quantitative RT-PCR. Microarray analysis identified 2 121 genes with a 2-fold change or above after TGF-β1 stimulation. 1 318 genes showed a greater than 2-fold increase and 761 genes were reduced 2 folds or more at mRNA levels, whereas a small portion of the total regulated genes （42 genes） displayed dynamically up- and down-regulated pattern. Genes were further segregated for early （peak at 5 min, 15 min and/or 2 h）, late （peak at 12 h and/or 24 h）, and sustained （2-fold change or above at five time points） temporal response groups according to the time of their peak expression level, Among 1 318 up-regulated genes, 333 genes （25.3%） responded rapidly to TGF-β1 and 159 genes （12.1%） responded in a sustained manner. Most genes （826, 62.6%） were regulated at 12 h or later. For the 761 down-regulated genes, numbers of early and late responsive genes were 335 （44%） and 267 （36.1%）, respectively. There were also 159 genes, 19.9% of total down-regulated genes, decreased at five time points treated by TGF-β1. The results suggested that the gene expressions of secreted phosphoprotein 1 （APP1） and Rho-associated coiled-coil forming kinase 2 （ROCK2） had the same trends as α-smooth muscle-actin, a marker of MF differentiation. In addition, the gene expression of potassium voltage-gated channel, Shal-related family and member 2 （KCND2） was up-regulated. Furthermore, it was found that endothelin 1 （EDN 1）, some complement components, NADPH oxidase 4 （NOX4） and NAD（P）H dehydrogenase, quinone 1 （NQO 1） might be involved in MF differentiation. Using microarrary technique, we confirmed some genes that have been identified by other techniques were implicated in MF differentiation and observed new genes involved in this process. Our results suggest that gene expression profiling study is helpful in identifying genes and pathways potentially involved in cell differentiation.
出处
《生理学报》
CAS
CSCD
北大核心
2006年第4期337-344,共8页
Acta Physiologica Sinica
基金
This work was supported by the National Basic Research Priorities Programme of China(No.2004CB518603)and Key Project of Basic Research from Shanghai Science and Technology Committee(No.05JC14038).
关键词
寡核苷酸芯片
外膜
成纤维细胞
分化
oligonucleotide arrays
adventitia
fibroblast
differentiation
