摘要
背景:氟牙症是长期摄入大量氟所导致的牙釉质发育障碍,病因复杂,其发病机制有待深入研究。目的:通过转录组测序技术筛选高氟干预成釉细胞与钙稳态相关的差异表达基因,并进一步探索氟斑牙形成的分子机制。方法:分别用浓度为0,0.4,0.8,1.6,3.2,6.4 mmol/L的NaF处理成釉细胞LS824,48,72 h,检测细胞形态、细胞活性与细胞内钙离子浓度。分别用浓度为0,1.6,3.2 mmol/L的NaF处理成釉细胞LS824 h,通过转录组测序筛选差异表达基因,并对差异表达基因进行验证。结果与结论:①处理24 h后,NaF浓度0,0.4,0.8 mmol/L组细胞生长状态良好,细胞的数量增多,细胞轮廓清晰;当NaF浓度≥1.6 mmol/L,随着NaF浓度的增加,细胞体积逐渐皱缩变小、细胞数量减少。处理48,72 h后,NaF浓度0,0.4 mmol/L组细胞数量增加,0.8,1.6,3.2 mmol/L组细胞数量逐渐减少,细胞形态变圆、变小,6.4 mmol/L组细胞皱缩变圆悬浮于培养基中,几乎无细胞贴壁。当NaF浓度相同时,处理24 h后LS8细胞的生长状态最佳。CCK-8检测结果显示,当NaF浓度相同时,随着处理时间的延长,细胞活性减弱;当处理时间相同时,随着NaF浓度的增加,细胞活性减弱。处理24 h后,随着NaF浓度的增加,细胞内钙离子浓度增加。②转录组测序分析发现参与调控细胞钙稳态的基因:Hsp90b1、Canx、Calr、Hspa5的表达显著上调(P<0.05),Cacna1a的表达显著下调(P<0.05),该结果得到了RT-qPCR检测的验证。③结果显示,NaF对LS8细胞增殖的抑制作用可能与细胞内Ca2+浓度异常增加有关,其机制可能由蛋白质加工合成通路Hsp90b1、Canx、Calr、Hspa5表达上调和钙信号通路Cacna1a表达下调所导致。
BACKGROUND:Fluorosis is a disorder of enamel development caused by long-term intake of large amounts of fluoride during enamel development.OBJECTIVE:To further explore the molecular mechanism of dental fluorosis formation by screening the differentially expressed genes associated with calcium homeostasis in ameloblasts by transcriptome sequencing technology.METHODS:LS8 cells were treated with 0,0.4,0.8,1.6,3.2 and 6.4 mmol/L sodium fluoride(NaF)for 24,48 and 72 hours to observe the effects of different concentrations of NaF on the morphology,cell activity and intracellular Ca2+concentration of LS8 cells.The differentially expressed genes were screened by transcriptome sequencing and validated.RESULTS AND CONCLUSION:After 24 hours of treatment,the cells treated with 0,0.4,and 0.8 mmol/L NaF were in good growth condition,with increased cell number and clear cell outline.When the NaF concentration was≥1.6 mmol/L,the cells were gradually shrunken and became smaller and the number of cells decreased with the increase of NaF concentration.After 48 and 72 hours of treatment,the number of cells increased in the 0,0.4 mmol/L NaF groups,while gradually decreased in the 0.8,1.6,3.2 mmol/L NaF groups,with rounded and smaller cell morphology.The cells in the 6.4 mmol/L NaF group were shrunken,rounded and suspended in the medium,with almost no adherent cells.When treated with the same concentration of NaF,LS8 cells were in optimal growth after 24 hours of treatment.Results from cell counting kit-8 assay showed that when treated with the same concentration of NaF,the cell activity decreased with the increase of treatment time;when the treatment time was the same,the cell activity decreased with the increase of NaF concentration.After 24 hours of treatment,the intracellular Ca2+concentration increased with the increase of NaF concentration.Transcriptome sequencing analysis identified genes involved in the regulation of cellular calcium homeostasis:Hsp90b1,Canx,Calr,and Hspa5 that were significantly upregulated(P<0.05)and Cacna1a that was significantly downregulated(P<0.05).To conclude,the inhibitory effect of NaF on LS8 cell proliferation may be related to the abnormal increase in intracellular Ca2+concentration,and the mechanism may be caused by the upregulation of the expression of protein processing and synthesis pathways Hsp90b1,Canx,Calr,and Hspa5 and the downregulation of the expression of calcium signaling pathway Cacna1a.
作者
黄婷
刘霞
王烛
陈霆
陈彬
白国辉
吴家媛
田源
Huang Ting;Liu Xia;Wang Zhu;Chen Ting;Chen Bin;Bai Guohui;Wu Jiayuan;Tian Yuan(Affiliated Stomatology Hospital of Zunyi Medical University,Zunyi 563000,Guizhou Province,China;School of Stomatology,Zunyi Medical University,Zunyi 563000,Guizhou Province,China)
出处
《中国组织工程研究》
CAS
北大核心
2024年第16期2481-2487,共7页
Chinese Journal of Tissue Engineering Research
基金
国家自然科学基金(81960198),项目负责人:田源
遵义市口腔疾病免疫防治及医用生物材料研发创新人才团队项目(遵市科人才[2022]1号),项目负责人:白国辉
遵义医科大学附属口腔医院口腔感染性与恶性疾病的病因与防治团队项目[遵市科合HZ字(2020)],项目负责人:吴家媛。
关键词
氟中毒
氟斑牙
成釉细胞
氟化钠
转录组测序
内质网应激
钙离子通道
钙离子探针
fluorosis
dental fluorosis
ameloblast
sodium fluoride
transcriptome sequencing
endoplasmic reticulum stress
calcium channel
calcium probe
