Senescence focused gene arrays in MSC under different oxygen pressures

Expression of senescence associated genes (N=84) was screened in cultures of mesenchymal stromal cells from human healthy donors (N=4) simultaneously isolated and expanded in different oxygen pressure (5% and19.95%). We hypothesized that higher oxygen pressure would increase the expression of genes involved in cellular senescence. RNA from passage 3 cultures at 70% confluence during growth log phase RNA (Qiagen RNeasy) was transcribed using Qiagen RT2 First Strand kit, followed by amplification using Qiagen RT2 Profiler PCR Array focused on cellular senescence and Qiagen SBR Green PCR master mix. Data analysis was performed using the RT2 PCR Profiler PCR Array Data Analysis software provided by Qiagen. Data analysis was based on the ΔΔCT method with normalization of the raw data to housekeeping genes.We found only four genes that were differentially expressed (CDK4, MYC, P53BP1 and SOD2) with 2 fold change (p value less than 0.05). The array did not identify a specific senescence pathway. The samples were from primary cultures and in a small number, therefore data require validation using larger number of donors.本研究在4名健康人类供者的间充质基质细胞（mesenchymal stromal cells, MSCs）培养物中筛选84个衰老相关基因（senescence associated genes, SAGs）的表达情况；上述细胞均在5%与19.95%两种不同氧分压条件下同步完成分离与扩增。本研究假设，更高的氧分压会上调细胞衰老相关基因的表达。 本研究提取对数生长期、汇合度达70%的第3代培养物的RNA（采用Qiagen RNeasy试剂盒），使用Qiagen RT2 First Strand试剂盒进行反转录，随后借助靶向细胞衰老的Qiagen RT2 Profiler PCR芯片与Qiagen SBR Green PCR预混液完成扩增。数据分析采用Qiagen官方提供的RT2 PCR芯片数据分析软件开展，基于ΔΔCT法，以持家基因对原始数据进行标准化处理。 本研究仅筛选出4个差异表达基因（CDK4、MYC、P53BP1与SOD2），其表达变化倍数为2倍，p值小于0.05。该PCR芯片未识别到特定的衰老相关通路。由于本研究样本均来自原代培养且样本量有限，因此所得数据需通过更大规模的供者样本进行验证。