Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes.
Storage Temperature Alters the Expression of Differentiation-Related Genes in Cultured Oral Keratinocytes.
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储存温度改变了培养的口服角质形成细胞中分化相关基因的表达。
DOI：
10.1371/journal.pone.0152526
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Pasovic L
中科院分区：
文献类型：
--
作者：
Utheim TP;Islam R;Fostad IG;Eidet JR;Sehic A;Olstad OK;Dartt DA;Messelt EB;Griffith M;Pasovic L
培养的人类口腔角质形成细胞 (HOK) 的储存可以将培养的移植物运输到世界各地的眼科诊所。在之前的一项研究中，发现与 4°C 和 37°C 相比，在 12°C 下保存一周的培养 HOK 在活力和形态方面更佳。为了更多地了解储存温度如何影响细胞表型，评估了 4°C、12°C 和 37°C 储存前后 HOK 的基因表达。培养的 HOK 在 HEPES 和碳酸氢钠缓冲的最低必需培养基中于 4°C、12°C 和 37°C 保存一周。分离总 RNA，使用 DNA 微阵列测定基因表达谱，并使用 Partek Genomics Suite 软件和 Ingenuity Pathway Analysis 进行分析。通过单向方差分析鉴定差异表达基因（倍数变化 > 1.5 且 P < 0.05）。使用 qPCR 验证关键基因。 4°C 和 12°C 下储存的培养物的基因表达与未储存的对照培养物接近。与其他组相比，保存在 37°C 下的培养物显示出基因表达的显着变化。与12℃相比，37℃下有2,981个基因差异表达。相比之下，未保存的对照细胞和 12°C 保存的细胞之间只有 67 个基因存在差异表达。 12°C 和 37°C 培养组在分化标记物的表达方面差异最显着。与 12°C 相比，37°C 时 Hedgehog 信号通路显着下调。与未保存的细胞相比，保存在 37°C 下的 HOK 培养物显示出比保存在 4°C 和 12°C 下培养的 HOK 更大的基因表达变化。在 37°C 下观察到的变化包括细胞向鳞状上皮特异性表型的分化。因此，不建议将培养的眼表移植物储存在 37°C 下。这是特别有趣的，因为 37°C 是细胞培养的标准孵化温度。
Storage of cultured human oral keratinocytes (HOK) allows for transportation of cultured transplants to eye clinics worldwide. In a previous study, one-week storage of cultured HOK was found to be superior with regard to viability and morphology at 12°C compared to 4°C and 37°C. To understand more of how storage temperature affects cell phenotype, gene expression of HOK before and after storage at 4°C, 12°C, and 37°C was assessed. Cultured HOK were stored in HEPES- and sodium bicarbonate-buffered Minimum Essential Medium at 4°C, 12°C, and 37°C for one week. Total RNA was isolated and the gene expression profile was determined using DNA microarrays and analyzed with Partek Genomics Suite software and Ingenuity Pathway Analysis. Differentially expressed genes (fold change > 1.5 and P < 0.05) were identified by one-way ANOVA. Key genes were validated using qPCR. Gene expression of cultures stored at 4°C and 12°C clustered close to the unstored control cultures. Cultures stored at 37°C displayed substantial change in gene expression compared to the other groups. In comparison with 12°C, 2,981 genes were differentially expressed at 37°C. In contrast, only 67 genes were differentially expressed between the unstored control and the cells stored at 12°C. The 12°C and 37°C culture groups differed most significantly with regard to the expression of differentiation markers. The Hedgehog signaling pathway was significantly downregulated at 37°C compared to 12°C. HOK cultures stored at 37°C showed considerably larger changes in gene expression compared to unstored cells than cultured HOK stored at 4°C and 12°C. The changes observed at 37°C consisted of differentiation of the cells towards a squamous epithelium-specific phenotype. Storing cultured ocular surface transplants at 37°C is therefore not recommended. This is particularly interesting as 37°C is the standard incubation temperature used for cell culture.