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

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.