Mahidol University, Faculty of Dentistry, Department of Oral Biology, Thailand.
Mahidol University, Faculty of Dentistry, Department of Pediatric Dentistry, Thailand.
J Appl Oral Sci. 2021 Sep 27;29:e20201074. doi: 10.1590/1678-7757-2020-1074. eCollection 2021.
Hyperglycemia, a major characteristic of diabetes, is considered to play a vital role in diabetic complications. High glucose levels have been found to inhibit the mineralization of dental pulp cells. However, gene expression associated with this phenomenon has not yet been reported. This is important for future dental therapeutic application.
Our study aimed to investigate the effect of high glucose levels on mineralization of human dental pulp-derived cells (hDPCs) and identify the genes involved.
hDPCs were cultured in mineralizing medium containing 25 or 5.5 mM D-glucose. On days 1 and 14, RNA was extracted and expression microarray performed. Then, differentially expressed genes (DEGs) were selected for further validation using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) method. Cells were fixed and stained with alizarin red on day 21 to detect the formation of mineralized nodules, which was further quantified by acetic acid extraction.
Comparisons between high-glucose and low-glucose conditions showed that on day 1, there were 72 significantly up-regulated and 75 down-regulated genes in the high-glucose condition. Moreover, 115 significantly up- and 292 down-regulated genes were identified in the high-glucose condition on day 14. DEGs were enriched in different GO terms and pathways, such as biological and cellular processes, metabolic pathways, cytokine-cytokine receptor interaction and AGE-RAGE signaling pathways. RT-qPCR results confirmed the significant expression of pyruvate dehydrogenase kinase 3 (PDK3), cyclin-dependent kinase 8 (CDK8), activating transcription factor 3 (ATF3), fibulin-7 (Fbln-7), hyaluronan synthase 1 (HAS1), interleukin 4 receptor (IL-4R) and apolipoprotein C1 (ApoC1).
The high-glucose condition significantly inhibited the mineralization of hDPCs. DEGs were identified, and interestingly, HAS1 and Fbln-7 genes may be involved in the glucose inhibitory effect on hDPC mineralization.
本研究旨在探讨高糖环境对人牙髓细胞（hDPCs）矿化的影响，并鉴定相关基因。
将 hDPCs 培养在含 25 或 5.5 mM D-葡萄糖的矿化培养基中。在第 1 天和第 14 天提取 RNA 并进行表达微阵列分析。然后，采用逆转录定量聚合酶链反应（RT-qPCR）方法对差异表达基因（DEGs）进行进一步验证。在第 21 天，细胞用茜素红固定和染色，以检测矿化结节的形成，并通过乙酸提取进一步定量。
高糖与低糖条件的比较显示，在第 1 天，高糖条件下有 72 个显著上调和 75 个下调基因；在第 14 天，高糖条件下有 115 个显著上调和 292 个下调基因。DEGs 在不同的 GO 术语和途径中富集，如生物和细胞过程、代谢途径、细胞因子-细胞因子受体相互作用和 AGE-RAGE 信号通路。RT-qPCR 结果证实了丙酮酸脱氢酶激酶 3（PDK3）、细胞周期蛋白依赖性激酶 8（CDK8）、激活转录因子 3（ATF3）、纤维连接蛋白-7（Fbln-7）、透明质酸合酶 1（HAS1）、白细胞介素 4 受体（IL-4R）和载脂蛋白 C1（ApoC1）的显著表达。
高糖环境显著抑制 hDPCs 的矿化。鉴定出差异表达基因，有趣的是，HAS1 和 Fbln-7 基因可能参与了葡萄糖对 hDPC 矿化的抑制作用。
