Utheim Tor Paaske, Salvanos Panagiotis, Utheim Øygunn Aass, Ræder Sten, Pasovic Lara, Olstad Ole Kristoffer, de la Paz Maria Fideliz, Sehic Amer
Department of Medical Biochemistry, Oslo University Hospital, Oslo 0407, Norway.
Department of Ophthalmology, Drammen Hospital, Vestre Viken Hospital Trust, Drammen 3004, Norway.
J Funct Biomater. 2016 Feb 18;7(1):4. doi: 10.3390/jfb7010004.
The aim of the present study was to investigate the molecular mechanisms underlying activation of cell death pathways using genome-wide transcriptional analysis in human limbal epithelial cell (HLEC) cultures following conventional hypothermic storage in Optisol-GS. Three-week HLEC cultures were stored in Optisol-GS for 2, 4, and 7 days at 4 °C. Partek Genomics Suite software v.6.15.0422, (Partec Inc., St. Louis, MO, USA) was used to identify genes that showed significantly different (P < 0.05) levels of expression following hypothermic storage compared to non-stored cell sheets. There were few changes in gene expression after 2 days of storage, but several genes were differently regulated following 4 and 7 days of storage. The histone-coding genes HIST1H3A and HIST4H4 were among the most upregulated genes following 4 and 7 days of hypothermic storage. Bioinformatic analysis suggested that these two genes are involved in a functional network highly associated with cell death, necrosis, and transcription of RNA. HDAC1, encoding histone deacetylase 1, was the most downregulated gene after 7 days of storage. Together with other downregulated genes, it is suggested that HDAC1 is involved in a regulating network significantly associated with cellular function and maintenance, differentiation of cells, and DNA repair. Our data suggest that the upregulated expression of histone-coding genes together with downregulated genes affecting cell differentiation and DNA repair may be responsible for increased cell death following hypothermic storage of cultured HLEC. In summary, our results demonstrated that a higher number of genes changed with increasing storage time. Moreover, in general, larger differences in absolute gene expression values were observed with increasing storage time. Further understanding of these molecular mechanisms is important for optimization of storage technology for limbal epithelial sheets.
本研究的目的是利用全基因组转录分析，研究人角膜缘上皮细胞（HLEC）培养物在Optisol - GS中进行常规低温保存后细胞死亡途径激活的分子机制。将3周龄的HLEC培养物在Optisol - GS中于4℃保存2、4和7天。使用Partek Genomics Suite软件v.6.15.0422（美国密苏里州圣路易斯市的Partec公司）来鉴定与未保存的细胞片相比，低温保存后表达水平有显著差异（P < 0.05）的基因。保存2天后基因表达变化很少，但保存4天和7天后有几个基因受到不同调控。组蛋白编码基因HIST1H3A和HIST4H4是低温保存4天和7天后上调最多的基因。生物信息学分析表明，这两个基因参与了一个与细胞死亡、坏死和RNA转录高度相关的功能网络。编码组蛋白脱乙酰酶1的HDAC1是保存7天后下调最多的基因。与其他下调基因一起，提示HDAC1参与了一个与细胞功能和维持、细胞分化以及DNA修复显著相关的调控网络。我们的数据表明，组蛋白编码基因的上调表达以及影响细胞分化和DNA修复的下调基因可能是培养的HLEC低温保存后细胞死亡增加的原因。总之，我们的结果表明，随着保存时间的增加，有更多的基因发生变化。此外，一般来说，随着保存时间的增加，绝对基因表达值的差异更大。进一步了解这些分子机制对于优化角膜缘上皮片的保存技术很重要。
