自噬在延期牙再植术后炎症性牙根吸收中的作用研究

Effects of autophagy in inflammatory root resorption after delayed tooth replantation

目的:观察大鼠延期牙再植术后自噬水平变化,探讨自噬在延期牙再植术后炎症性牙根吸收中的作用。方法:选取24只4周龄SD大鼠,拔除双侧上颌第一磨牙,干燥30 min后植回牙槽窝,术后1、3、7、28 d取材,染色观察炎症浸润情况、牙根表面吸收情况、肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)等炎症因子表达及LC3B和Beclin-1等自噬相关蛋白的表达情况。使用1μg/mL脂多糖(LPS)处理人牙周膜成纤维细胞(hPDLFs),细胞免疫荧光染色观察不同时间点LC3B和Beclin-1的表达,LPS中分别添加自噬促进剂雷帕霉素和自噬抑制剂氯喹,实时定量RT-PCR检测炎症和自噬相关蛋白的基因转录水平。结果:牙再植术后第1、3、7天牙周组织中炎症细胞浸润、炎症因子分泌逐渐增加,第7天时炎症性牙根吸收明显,第28天炎症明显好转。自噬相关蛋白表达在术后第1天明显上升,第3、7天表达下降,第28天再次增多。体外细胞免疫荧光染色结果显示自噬相关蛋白在LPS刺激24 h时被激活,72 h时被抑制,体外细胞与体内组织自噬相关蛋白表达趋势一致。实时定量RT-PCR结果显示,在LPS处理的hPDLFs中加入自噬促进剂后,炎症因子表达明显减少。结论:自噬参与延期牙再植术后炎症性牙根吸收的发生发展,并可能起保护作用。

Objective:To observe the changes of autophagy levels after delayed tooth replantation in rats and investigate the effect of autophagy in inflammatory root resorption after delayed tooth replantation.Methods:Twenty-four 4-week-old SD rats were divided into different groups.Maxillary first molars were extracted,dried for 30 minutes and then replanted into the alveolar sockets.Rats were sacrificed on day 1,3,7 and 28 after surgery,the inflammation infiltration situation,root resorption situation,expression of inflammatory cytokines,TNF-αand IL-1β,and expression of autophagy proteins LC3B and Beclin-1 were detected by tissue staining.Immunofluorescence staining was performed on human periodontal ligament fibroblasts(hPDLFs)treated with LPS to detect the expression of LC3B and Beclin-1 at different time points.Meanwhile,the inflammation and autophagy-related gene expression in hPDLFs treated with LPS and autophagy promoter(Rapamycin)or autophagy inhibitor(chloroquine)was examined through real-time RT-PCR.Results:Massive inflammatory cell infiltration and inflammatory cytokines TNF-αand IL-1βaround the root were observed on the 1st and 3rd days after surgery.On the 7th day,inflammatory infiltration level increased gradually and increased secretion of TNF-αand IL-1βwas detected through immunohistochemical staining.The TRAP staining result showed obvious inflammatory resorption on the 7th day.The inflammation alleviated on the 28th day.During this process,autophagy proteins LC3B and Beclin-1 increased significantly on the 1st day after surgery,then declined on the 3rd and 7th day and increased again on the 28th day.In vitro experiments,autophagy proteins LC3B and Beclin-1 were activated in hPDLFs after LPS 24 h stimulation,while inhibited after 72 h LPS stimulation.The trends of autophagy proteins in vitro were consistent with the in vivo results.Expression of inflammation genes were significantly reduced in hPDLFs treated with autophagy promoter and LPS.Conclusions:Autophagy participates in the development of root inflammatory resorption after delayed tooth replantation,and may play a protective role.