降钙素抑制NF-κB活性促牙周膜干细胞成骨分化的作用机制研究

Periodontitis-caused alveolar bone lesion and absorption is the leading cause of tooth loss. Recently, we reported for the first time that calcitonin (CT) in gingival crevicular fluid negatively correlated with the clinical indicators of periodontitis, which was found to induce osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Our results revealed that CT plays a key role in alveolar bone regeneration by accelerating osteogenesis of PDLSCs. In further study, we found that CT over-expression could dramatically inhibit NF-κB transcription activity in PDLSCs. Moreover, NF-κB inhibitor treatment also induced PDLSCs osteogenic differentiation. Accordingly, we propose that CT could promote PDLSCs osteogenic differentiation by inhibiting NF-κB transcription activity. In order to verify this hypothesis, we intend to perform experiments including luciferase reporter assay, co-immunoprecipitation and fluorescence resonance energy transfer, to confirm CT could inhibit the transcription activity of NF-κB by up-regulating the expression of transcription factor ICER. Moreover, we aim to determine the role of CT/NF-κB pathway in promoting osteogenic differentiation of PDLSCs via in vitro techniques. Also, rat periodontitis model will be established to further illustrate the protective effects of CT/NF-κB pathway in alveolar bone injury induced by periodontitis. Our project could provide novel intervention targets for the alveolar bone regeneration in clinic.

牙周炎导致的牙槽骨损伤吸收是牙齿丧失最主要的原因。本课题组近来首次报道了降钙素（CT）在患者龈沟液中的表达水平与牙周炎临床指标呈负相关，且CT可诱导牙周膜干细胞（PDLSC）成骨分化，提示CT促进PDLSC成骨分化可能在牙周炎所致的牙槽骨损伤后修复过程中发挥关键作用。后续研究我们发现过表达CT可显著抑制PDLSC中NF-κB转录活性，鉴于NF-κB抑制剂处理可诱导PDLSC成骨分化，由此我们提出了"CT通过抑制NF-κB转录活性促PDLSC成骨分化"的假说。为验证该假说，本项目拟通过荧光素酶活性报告、免疫共沉淀及荧光共振能量转移等实验明确CT上调反式因子ICER的表达，继而结合NF-κB并抑制其活性的具体机制；通过细胞功能实验明确CT/NF-κB通路调控PDLSC成骨分化的作用；此外，将建立大鼠牙周炎模型观察CT/NF-κB通路在牙周炎引发牙槽骨损伤中的作用并探讨其作为治疗靶点的潜在可能。

牙周炎导致的牙槽骨损伤吸收是牙齿丧失最主要的原因。本课题组近来首次报道了降钙素（CT）在患者龈沟液中的表达水平与牙周炎临床指标呈负相关，且CT可诱导牙周膜干细胞（PDLSC）成骨分化，提示CT促进PDLSC成骨分化可能在牙周炎所致的牙槽骨损伤后修复过程中发挥关键作用。后续研究我们发现过表达CT可显著抑制PDLSC中NF-κB转录活性，鉴于NF-κB抑制剂处理可诱导PDLSC成骨分化，由此我们提出了"CT通过抑制NF-κB转录活性促PDLSC成骨分化"的假说。在本研究中，我们通过腺病毒介导的转基因在PDLSCs中过表达CT后，在不同时间天数使用茜素红染色，对比观察细胞钙沉积情况。结果提示CT处理PDLSCs后7天表现的成骨分化趋势，14天时成骨分化明显。通过腺病毒介导的转基因在PDLSCs中过表达CT后，通过Western blot检测CREB/pCREB/ICER表达水平。Western blot结果显示，PDLSCs感染Ad.CT后，CREB表达在1、3、7、14天均维持在高水平，且均显著高于空病毒感染组，同时各时间点的CREB表达水平没有显著差异。pCREB表达水平也均较空病毒感染组显著提高，但我们注意到pCREB的表达高峰出现在第3天。茜素红染色结果显示7天及14天时，与空病毒感染组相比过表达CT组钙化显著。OPN的Western blot结果同样显示7天及14天时，过表达CT组中ICER的蛋白表达水平显著高于空病毒感染组。通过EMSA发现，过表达CT后能够显著抑制NF-κB的转录活性。构建大鼠牙周炎模型后给与CT干预。结果显示，建模组的CREB、pCREB、ICER均较正常对照组显著下降，而建模后给与CT干预，尽管CREB、pCREB、ICER的表达水平仍然低于正常对照组，但是较单纯建模组显著上升。综上所述，我们认为CT可通过促进CREB磷酸化的途径激活CREB的转录活性，进而促进ICER表达。ICER具有抑制NF-κB转录活性的作用，从而诱导PDLSCs成骨分化。