【作者】 祁胜财；

【导师】 朱声荣；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 人牙髓细胞（humandentalpulpcells，hDPCs）在受到适当的刺激后，可通过增殖、迁移和成牙本质细胞分化而形成修复性牙本质。牙髓对损伤的应答和修复性牙本质的形成是一个复杂的生物学过程，涉及牙髓细胞（Dentalpulpcells，DPCs）的增殖、迁移、成牙本质细胞分化和牙本质基质的形成与矿化。然而，hDPCs增殖和成牙本质细胞分化的机制仍不清楚。高迁移率族蛋白B1（High-mobilitygroupbox1protein，HMGB1）属高迁移率族蛋白家族成员，是一种DNA结合蛋白，能适当的调节转录因子和DNA重组及修复。还具有多种生物学效应，HMGB1还可作为细胞外信号分子，调节炎症反应和组织再生。有研究发现在心肌梗死后的心肌组织再生中，HMGB1能诱导心肌前体细胞的增殖和分化；HMGB1能以自分泌或旁分泌的形式参与骨骼肌损伤后的组织再生；HMGB1可通过促进皮肤成纤维细胞的增殖和迁移，促进皮肤伤口的愈合；HMGB1还在大鼠神经前体细胞的增殖和向神经细胞分化中发挥重要作用。牙齿发育过程中，HMGB1在矿化区域中的成釉细胞和成牙本质细胞中高表达，提示HMGB1可能参与了牙齿的矿化。因此，探讨HMGB1与hDPCs的增殖、迁移和分化的关系具有重要意义。目的检测HMGB1在人牙髓组织和牙髓细胞中的表达，并采用体外培养的hDPCs为实验模型，以HMGB1作为细胞外刺激因子，研究HMGB1与hDPCs的增殖、迁移和分化的关系。方法采用组织块法体外原代培养hDPCs；免疫组化法和免疫荧光法分别检测HMGB1在牙髓组织和牙髓细胞中的表达情况，流式细胞术定量检测hDPCsHMGB1的表达率；以不同浓度的HMGB1作为细胞外刺激因子，通过CCK-8试剂盒检测HMGB1对hDPCs的增殖效应，并通过细胞划痕实验初步探究HMGB1对hDPCs迁移能力的影响；hDPCs矿化诱导后，检测牙髓细胞碱性磷酸酶活性及矿化结节形成情况，荧光定量PCR和WesternBlot检测牙本质涎磷蛋白（Dentinsialophosphoprotein，DSPP）、牙本质基质蛋白（Dentinmatrixprotein-1，DMP-1）、碱性磷酸酶（Alkalinephosphatase，ALP）以及HMGB1和晚期糖基化终产物受体（Receptorforadvancedglycationendproducts，RAGE）的表达情况，采用细胞免疫荧光和WesternBlot分别检测矿化过程中不同时间点HMGB1在hDPCs细胞核和细胞质的分布以及蛋白表达水平，采用ELISA试剂盒检测不同时间点上清中HMGB1的含量；在hDPCs的矿化过程中，以适宜浓度HMGB1刺激细胞后，荧光定量PCR和WesternBlot检测DSPP、DMP-1、ALP以及RAGE的表达情况。结果组织块法原代培养hDPCs切实可行；HMGB1在人牙髓组织和hDPCs细胞核中表达，适宜浓度的HMGB1可促进hDPCs增殖和迁移；在hDPCs的矿化诱导中，HMGB1的mRNA水平上调，且从细胞核转位至细胞质，并主动分泌至细胞外；HMGB1可促进矿化结节的形成，并上调hDPCs的ALPase活性，以及DSPP、DMP-1、ALP及RAGE的mRNA和蛋白质水平。结论本研究发现HMGB1在人牙髓组织和约98%的hDPCs细胞核中表达。HMGB1与hDPCs的成牙本质细胞分化相关，在矿化过程中，HMGB1可从hDPCs细胞核转位至细胞质，并分泌至细胞外。适宜浓度HMGB1刺激hDPCs后，可能通过受体RAGE，促进细胞增殖、迁移和分化。更多还原

【Abstract】 Background and Objective: Human dental pulp cells (hDPCs) can proliferate and differentiate into odontoblasts thus creating reparative dentine in response to the appropriate stimuli. The response to stimuli and formation of reparative dentine of pulp tissue is a complex biological process, which involves the migration, proliferation and differentiation of hDPCs into odontoblast/odontoblasts-like cells. However, the precise mechanisms underlying the proliferation and differentiation of hDPCs remain unclear. High-mobility group box1protein (HMGB1) belongs to a family of high-mobility group nuclear proteins, which is a DNA-binding protein that has nuclear effects, including proper transcriptional regulation and DNA recombination and repair. In addition to its nuclear role, HMGB1functions as an extracellular signaling molecule that regulates both inflammation and regeneration. According to previous studies, HMGB1induced cardiac progenitor cell proliferation and differentiation in myocardial regeneration; HMGB1promoted the wound healing through inducing fibroblast cell proliferation and migration; HMGB1took part in the skeletal muscle regeneration, modulating in an autocrine or paracrine manner; and HMGB1played an important role in the proliferation and neuronal differentiation of mouse neuronal progenitor cells by binding with receptor for advance glycation end products (RAGE). A recent study showed that the expression of HMGB1was particularly high in ameloblasts and odontoblasts at regions of ongoing mineralization, which indicated that HMGB1participates in tooth mineralization. Therefore, it is meaningful to research the biological effects of HMGB1on hDPCs. The aims of this study were to investigate the expression of high-mobility group box1(HMGB1) in human dental pulp and the biological effects of HMGB1on proliferation, migration and odontoblastic differentiation of hDPCs.Methodology: Human dental pulp cells were cultivated from pulp connective tissue explants. Cells between the third and sixth passages were used in this study. Immunohistochemical assay, immunoflurescence staining and flow cytometric analysis were used to detect the expression of HMGB1in the human dental pulp and hDPCs, respectively. After culturing human primary hDPCs in the presence of HMGB1with different doses, the proliferation of hDPCs was examined by CCK-8and the migration of hDPCs was evaluated using Scratch Test. Odontoblastic differentiation of hDPCs was determined using alkaline phosphatase (ALP) activity assay and mineralized nodule formation. Important mineralization-related genes such as ALP, dentin sialophosphoprotein (DSPP) and dentin matrix protein-1(DMP-1) were determined by real-time polymerase Chain reaction and Western Blot. Messenger RNA and protein levels of HMGB1and RAGE were also detected. immunoflurescence staining and Western Blot were used to detect the expression of HMGB1on the nuclei and the cytoplast. Meanwhile, the protein level of HMGB1in the supernatants was quantified using ELISA analysis on day0,3,7,11and14. Real-time polymerase Chain reaction and Western blot analysis were performed to determine the difference of expressions of DMP-1, DSP and ALP with or without the presence of exogenous HMGB1. Mineralized nodule formation, messenger RNA and protein levels of RAGE were also detected.Results:hDPCs culticated from pulp connective tissue explants was available. HMGB1was found in human dental pulp and in the nuclei of hDPCs, and was expressed on about98%of hDPCs. The appropriate concentration of HMGB1promotes the proliferation and migration of hDPCs. During hDPC odontoblastic differentiation, the mRNA level of HMGB1was up-regulated and translocated from the nuclei to the cytoplasm and then secreted out from hDPCs. Exogenous HMGB1promoted hDPC mineralized nodule formation. It up-regulated the activity of ALP and the mRNA and protein levels of DMP-1, ALP, DSPP, and RAGE of hDPCs.Conclusion:Taken together, these results suggest that hDPCs can be cultivated preferably from tissue explants in vitro. HMGB1was found in human dental pulp and in the nuclei of hDPCs, and was expressed on about98%of hDPCs. The appropriate concentration of HMGB1promotes the proliferation and migration of hDPCs. During hDPC odontoblastic differentiation, HMGB1was translocated from the nuclei to the cytoplasm and then actively secreted out from hDPCs. The appropriate concentration of HMGB1promotes the proliferation, migration and odontoblastic differentiation of hDPCs which maybe depend on RAGE.更多还原