T型钙通道对人牙囊细胞成骨分化的影响  

Effect of T-type Calcium Channel on Osteogenic Differentiation of Human Dental Follicle Cells

作　　者：杨晶晶 左东川 谢沂航 蔡欣 袁小平 曾锦 YANG Jingjing;ZUO Dongchuan;XIE Yihang;CAI Xin;YUAN Xiaoping;ZENG Jin(Department of Orthodontics, the Affiliated Stomatology Hospital of Southwest Medical University, Luzhou 646000, China;Laboratory of Oral and Maxillofacial Reconstruction and Regeneration, Southwest Medical University, Luzhou 646000, China;Institute of Cardiovascular Medicine, Key Laboratory of Electrophysiology of Ministry of Education, Southwest Medical University, Luzhou 646000, China.)

机构地区：[1]西南医科大学附属口腔医院正畸科,四川泸州646000 [2]西南医科大学口颌面修复重建和再生实验室,四川泸州646000 [3]西南医科大学电生理学教育部重点实验室,心血管医学研究所,四川泸州646000

出　　处：《口腔医学研究》2021年第10期900-905,共6页Journal of Oral Science Research

基　　金：泸州市-西南医科大学联合项目(编号:2020LZXNYDJ05);四川省泸州市科技局应用基础研究项目(编号:2019-JYJ-55)。

摘　　要：目的:探讨Ca^(2+)对人牙囊细胞(hDFCs)成骨分化的影响及其分子机制。方法:分离培养hDFCs,免疫荧光染色及流式细胞术检测hDFCs的来源。反转录聚合酶链反应(RT-PCR)检测L型钙通道、T型钙通道、三磷酸肌醇受体以及雷诺定受体在hDFCs的表达及-成骨分化相关基因RUNX相关转录因子2(runt-related transcription factor 2,RUNX2)、骨钙素(osteocalcin,OCN)的表达。茜素红染色分别检测移除细胞外Ca^(2+)、细胞内Ca^(2+)以及应用电压门控钙通道及Ca^(2+)/CaMKⅡ信号通路特异阻断剂后,hDFCs的成骨水平。结果:移除细胞外Ca^(2+)或细胞内Ca^(2+)后,hDFCs成骨能力显著降低。RT-PCR结果显示,hDFCs表达L型钙通道、T型钙通道、三磷酸肌醇受体以及雷诺定受体。L型钙通道阻断剂Nifedipine对细胞成骨能力没有影响,而T型钙通道阻断剂Amlioride能够明显抑制细胞的成骨水平。提高细胞外Ca^(2+)的浓度(5 mmol/L)能够促进hDFCs成骨分化,应用CaMKⅡ特异性阻断剂KN-93(10μmol/L)后,Ca^(2+)对hDFCs成骨分化的促进作用明显受到抑制。结论:T型钙通道介导的钙离子内流参与调控hDFCs的成骨分化。Objective:To investigate the effect of Ca^(2+)on osteogenic differentiation of human dental follicle cells(hDFCs)and to explore its molecular mechanism.Methods:hDFCs were isolated and cultured.Its source was verified by flow cytometry and immunofluorescence staining.The gene expression of L-type calcium channel,T-type calcium channel,inositol triphosphate receptor,and ryanodine receptor of hDFCs and mRNA expression of osteogenic differentiation related gene were detected by reverse transcription polymerase chain reaction(RT-PCR).Alizarin red staining was used to evaluate the formation of calcium nodules after the removal of extracellular Ca^(2+),intracellular Ca^(2+),and the application of voltage-gated calcium channel specific blockers.Results:The osteogenic ability of hDFCs was significantly decreased after removal of extracellular Ca^(2+)or intracellular Ca^(2+).RT-PCR results showed that hDFCs expressed L-type calcium channel,T-type calcium channel,inositol triphosphate receptor,and ryanodine receptor.Nifedipine,an L-type calcium channel blocker,had no effect on the osteogenic mineralization of the cells,while amlioride,a T-type calcium channel blockers significantly inhibited the osteogenic mineralization of the cells.Elevation of extracellular Ca^(2+)concentration(5 mmol/L)significantly promoted osteogenic differentiation of hDFCs,and the effect was inhibited by application of KN-93(10μmol/L,a Ca^(2+)/CaMKⅡsignaling pathway specific blocker).Conclusion:T-type calcium channel mediated calcium influx is involved in the osteogenic differentiation of hDFCs.

关 键 词：人牙囊细胞 钙离子 T型钙通道 成骨分化 

分 类 号：R73[医药卫生—肿瘤]