Supplementary Material for: Microarray Analysis of Irradiated Growth Plate Zones following Laser Microdissection Shows Later Importance of Differentially Expressed Genes during Radiorecovery

<i>Purpose:</i> Potential targets for selective radiorecovery modulation were investigated via the identification of late upregulated genes and pathways during growth plate chondrocyte recovery. <i>Methods and Materials:</i> Three groups of six 5-week-old male Sprague-Dawley rats underwent fractionated irradiation to the right tibiae over 5 days totaling 17.5 Gy and were then killed at 7, 11, and 16 days following the first radiotherapy fraction. The growth plates were collected from the proximal tibiae bilaterally and subsequently underwent laser microdissection to separate reserve, perichondral, proliferative, and hypertrophic zones. Differential gene expression was analyzed between irradiated right and nonirradiated left tibiae using RAE230 2.0 GeneChip microarray, compared between zones and time points, and subjected to functional pathway cluster analysis with real-time polymerase chain reaction (PCR) to confirm selected results. <i>Results:</i> The reserve zone showed the greatest number of differentially expressed genes and enriched pathways: 259 and 134, respectively. Differentially expressed genes included: Timp3, Gpx1, Gas6, Notch2, VEGF, and HIF-1. Enriched pathways included the developmental processes of regeneration, antiapoptosis, developmental growth, tissue regeneration, mesenchymal cell proliferation, negative regulation of immune response, and determination of symmetry. The reserve zone late upregulation of genes was validated using real-time PCR for Mgp, Gas6, and Eef1a1. <i>Conclusions:</i> A significant difference in late upregulated genes between growth plate zones exists. The reserve zone shows the greatest change, containing a 10-fold increase in the total number of genes differentially expressed between days 7 and 16. These findings suggest that reserve zone chondrocytes may play a later role in growth plate recovery response following irradiation.

研究目的：通过鉴定生长板软骨细胞恢复过程中晚期上调的基因与通路，探究选择性放射恢复调控的潜在作用靶点。材料与方法：选取3组、每组6只5周龄雄性Sprague-Dawley（斯普拉-道来）大鼠，对其右侧胫骨进行为期5天的分次照射，总照射剂量达17.5 Gy；分别于首次放疗分割后的第7、11、16天处死大鼠。采集双侧胫骨近端的生长板，随后通过激光显微切割（laser microdissection）分离储备区、软骨周区、增殖区与肥大区。采用RAE230 2.0型基因芯片微阵列（RAE230 2.0 GeneChip microarray）分析照射侧右侧胫骨与非照射侧左侧胫骨的差异基因表达情况，对比不同分区与不同时间点的基因表达差异，同时开展功能通路聚类分析，并通过实时聚合酶链反应（real-time polymerase chain reaction, PCR）对筛选得到的结果进行验证。结果：储备区的差异表达基因与富集通路数量最多，分别为259个与134个。筛选得到的差异表达基因包括Timp3、Gpx1、Gas6、Notch2、血管内皮生长因子（vascular endothelial growth factor, VEGF）以及缺氧诱导因子-1（hypoxia-inducible factor 1, HIF-1）。富集的通路涵盖再生、抗凋亡、发育性生长、组织再生、间充质细胞增殖、免疫应答负调控以及对称性确定等发育相关生物学过程。通过实时聚合酶链反应（PCR）验证了储备区基因的晚期上调表达特征，其中针对Mgp、Gas6及Eef1a1三个基因的验证结果与芯片分析结果一致。结论：生长板不同分区的晚期上调基因表达存在显著差异。其中储备区的变化最为显著，第7天至第16天之间的差异表达基因总数增加了10倍。上述研究结果表明，储备区软骨细胞可能在照射后生长板的恢复应答过程中发挥晚期调控作用。