Table_3_Gene Expression Profiles Reveal Extracellular Matrix and Inflammatory Signaling in Radiation-Induced Premature Differentiation of Human Fibroblast in vitro.XLSX

PurposeFibroblasts are considered to play a major role in the development of fibrotic reaction after radiotherapy and premature radiation-induced differentiation has been proposed as a cellular basis. The purpose was to relate gene expression profiles to radiation-induced phenotypic changes of human skin fibroblasts relevant for radiogenic fibrosis.
Materials and MethodsExponentially growing or confluent human skin fibroblast strains were irradiated in vitro with 1–3 fractions of 4 Gy X-rays. The differentiated phenotype was detected by cytomorphological scoring and immunofluorescence microscopy. Microarray analysis was performed on Human Genome U133 plus2.0 microarrays (Affymetrix) with JMP Genomics software, and pathway analysis with Reactome R-package. The expression levels and kinetics of selected genes were validated with quantitative real-time PCR (qPCR) and Western blotting.
ResultsIrradiation of exponentially growing fibroblast with 1 × 4 Gy resulted in phenotypic differentiation over a 5-day period. This was accompanied by downregulation of cell cycle-related genes and upregulation of collagen and other extracellular matrix (ECM)-related genes. Pathway analysis confirmed inactivation of proliferation and upregulation of ECM- and glycosaminoglycan (GAG)-related pathways. Furthermore, pathways related to inflammatory reactions were upregulated, and potential induction and signaling mechanisms were identified. Fractionated irradiation (3 × 4 Gy) of confluent cultures according to a previously published protocol for predicting the risk of fibrosis after radiotherapy showed similar downregulation but differences in upregulated genes and pathways.
ConclusionGene expression profiles after irradiation of exponentially growing cells were related to radiation-induced differentiation and inflammatory reactions, and potential signaling mechanisms. Upregulated pathways by different irradiation protocols may reflect different aspects of the fibrogenic process thus providing a model system for further hypothesis-based studies of radiation-induced fibrogenesis.

研究目的：成纤维细胞（Fibroblasts）被认为在放疗后纤维化反应的发生发展中发挥关键作用，而辐射诱导的过早分化已被提出作为该过程的细胞基础。本研究旨在探究与放射性纤维化相关的人类皮肤成纤维细胞的辐射诱导表型变化，及其与基因表达谱之间的关联。
材料与方法：将处于指数生长期或汇合期的人类皮肤成纤维细胞株置于体外，接受1~3次分割剂量的4Gy X射线照射。通过细胞形态学评分与免疫荧光显微镜检测，鉴定细胞的分化表型。采用Affymetrix人类基因组U133 plus2.0微阵列芯片（Human Genome U133 plus2.0 microarrays）结合JMP Genomics软件开展微阵列分析，并通过Reactome R-package进行通路分析。选取目标基因的表达水平及其动力学特征，通过实时荧光定量PCR（quantitative real-time PCR，qPCR）与蛋白质印迹法（Western blotting）进行验证。
结果：对指数生长期的成纤维细胞给予单次4Gy照射后，其表型分化过程持续5天。该过程伴随细胞周期相关基因的下调，以及胶原蛋白与其他细胞外基质（extracellular matrix，ECM）相关基因的上调。通路分析结果证实，细胞增殖通路被抑制，而ECM与糖胺聚糖（glycosaminoglycan，GAG）相关通路出现显著上调。此外，炎症反应相关通路亦被激活，本研究还鉴定出潜在的诱导机制与信号传导通路。按照既往发布的放疗后纤维化风险预测方案，对汇合期细胞给予3次分割剂量的4Gy照射，其细胞周期相关基因的下调趋势与单次照射组相似，但上调基因及通路存在明显差异。
结论：指数生长期细胞经辐射后的基因表达谱，与辐射诱导的分化、炎症反应及潜在信号传导机制密切相关。不同照射方案所激活的上调通路，可能反映纤维化发生过程的不同层面，因此本研究可为辐射诱导纤维化的后续假说驱动型研究提供理想的模型系统。