【作者】 李莉；

【导师】 骆凯；

【作者基本信息】 福建医科大学 ， 口腔医学（专业学位）， 2024， 博士

【摘要】 第一部分颌骨与股骨成骨细胞影响骨髓巨噬细胞表型和功能的体内外研究研究背景和目的在颌面部骨缺损修复重建中,长骨来源的移植物因其足够的骨量而经常被作为骨修复的供体移植物。已有研究表明,颅颌面来源的骨移植物具有更好的临床疗效,对颌面部骨缺损的修复成功率更高。不同骨位点的自体骨移植对骨移植的预后有不同的影响,其潜在机制尚未被完全阐明。骨移植后骨组织的再生过程涉及供受体部位不同类型细胞间的相互作用。成骨细胞(osteoblasts,OBs)和巨噬细胞(macrophages,M(?))作为参与骨再生修复的重要细胞,M(?)对OBs的作用已被广泛研究探讨,而OBs对M(?)的表型和功能的影响还鲜有报道。本实验将就颌骨和股骨来源的成骨细胞(osteoblats from the mandible and femur,MOBs and FOBs)对M(?)的表型和功能调节的差异性进行分析比较,研究结果将有助于进一步阐明颌面部骨重建位点特异性差异的潜在机制。研究方法体外培养颌骨、股骨成骨细胞及骨髓来源巨噬细胞(bone marrow-derived macrophages,BMDMs)。采用大鼠颅骨缺损模型,比较MOBs和FOBs对颅骨缺损再生修复的效果,以及骨缺损再生过程不同时间点(术后3、7、28天)M(?)的动态变化。收集MOBs和FOBs条件培养基(conditioned media,CM)培养BMDMs,比较不同位点来源的MOB-CM和FOB-CM对BMDMs活力、凋亡水平、迁移能力、吞噬功能和细胞极化的影响,以及对定向诱导分化后的M1及M2-BMDMs的作用。研究结果Micro-CT及组织学染色结果显示MOBs和FOBs均能够促进颅骨缺损的再生修复,其中MOBs的再生效果优于FOBs。免疫组化染色结果显示,MOBs可明显影响颅骨再生过程中M(?)细胞极化标志物和炎症相关因子的表达。术后3天,MOBs组M1-M(?)极化标志物i NOS阳性细胞明显较多,而M2-M(?)极化标志物Arg1阳性细胞则较少,IL-1β和IL-10的表达也均有明显增加;术后第7天,MOBs组的i NOS阳性细胞数量及IL-1β的表达均明显减少,而Arg1阳性细胞数量和IL-10的表达则均明显增加;术后28天MOBs组的Arg1、IL-1β和IL-10的表达仍较为明显。细胞实验结果显示,MOB-CM和FOB-CM对BMDMs的活力没有明显的作用,可轻微抑制BMDMs的凋亡,明显促进BMDMs的吞噬、迁移及产生NO的能力并抑制BMDMs的破骨分化。其中,MOB-CM对BMDMs的作用较FOB-CM明显。MOB-CM和FOB-CM均可促进BMDMs表达极化标志物i NOS和Arg1,也可增加M1-M(?)相关细胞因子和IL-10的表达,且MOB-CM的作用较为明显。对于定向诱导分化的M1及M2-BMDMs,MOB-CM和FOB-CM均能促进M1-BMDMs中Arg1和成骨相关细胞因子的表达,抑制促炎因子的表达,利于细胞向M2型的转换;但MOB-CM和FOB-CM对M2-BMDMs的影响则十分有限。结论MOBs和FOBs均可实现大鼠颅骨缺损的再生修复,该作用存在位点特异性,MOBs的再生效果要优于FOBs。MOBs和FOBs可影响BMDMs的表型和功能,其中MOBs的作用较为明显,更有利于形成骨组织再生的免疫微环境。第二部分炎症微环境中颌骨与股骨成骨细胞对骨髓来源巨噬细胞的作用及机制研究研究目的牙周炎是由牙周致病菌引发的慢性感染性疾病。牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)作为主要的牙周致病菌,与牙周炎的发生发展密切相关,其主要的毒力因子为脂多糖(Lipopolysaccharide,LPS)。研究业已证实Pg-LPS不仅在牙周炎所致牙槽骨破坏中发挥重要的作用,还可通过血液循环扩散至全身各脏器,进而影响全身健康。本课题第一部分研究发现不同位点成骨细胞对BMDMs的作用存在位点差异性,本部分研究拟进一步探究MOBs和FOBs在Pg-LPS所致炎症微环境下对BMDMs作用的差异及其机制,结果将为明确OBs如何通过M(?)影响牙周炎的进展及牙周炎与全身性骨代谢异常相关疾病之间的潜在联系提供实验依据。研究方法在Pg-LPS作用下,利用Transwell小室将MOBs及FOBs分别与BMDMs间接共培养,通过Western Blot、高通量全转录组测序、RT-qPCR等检测方法,探究MOBs和FOBs在Pg-LPS所致炎症微环境中对BMDMs表型和功能的作用及其机制。研究结果MOBs和FOBs在Pg-LPS所致炎症微环境中,均能降低BMDMs的STAT1磷酸化水平,从而显著抑制促炎因子IL-1β和TNF-α的表达。同时,它们还能通过上调STAT6磷酸化水平,促进M2-M(?)标记物Arg1的表达。此外,Pg-LPS作用下,FOBs促进BMDMs向M2-M(?)极化的作用更明显。结论在Pg-LPS作用下,MOBs和FOBs能够降低BMDMs的炎症反应,并诱导其向M2-M(?)极化,其中FOBs的作用更为明显。更多还原

【Abstract】 Part One:In vivo and in vitro studies on the modulation of bone marrow-derived macrophages phenotype and function via mandible and femur osteoblastsObjectiveIt’s well known that long bone grafts are often used as donor grafts in the repair and reconstruction of maxillofacial bone defect in terms of their sufficient bone volume.Studies have shown that craniomaxillofacial bone grafts have better clinical outcomes and higher success rates in the repair of maxillofacial bone defects.Autologous bone grafts from different bone sites have various effects on the prognosis of bone grafts,and the underlying mechanisms have not been completely elucidated.The regenerative process after bone transplantation involves interactions between different types of cells at the donor-recipient site.Osteoblasts(OBs)and macrophages(M(?)),as key cells involved in bone regeneration and repair,the effects of M(?)on OBs have been widely studied,whereas the effects of OBs on the phenotype and function of M(?)have rarely been reported.In this experiment,we are going to analyze and compare the differences in the phenotypic and functional modulation of M(?)by osteoblasts from the mandible and femur(OBs and FOBs),and the results of this study will contribute to further elucidate the potential mechanisms underlying the site-specific differences in maxillofacial bone reconstruction.MethodsMandible and femur osteoblasts and bone marrow-derived macrophages(BMDMs)were cultured in vitro.A rat cranial bone defect model was applied to compare the effects of MOBs and FOBs on the regeneration of cranial bone defects and the dynamic changes of M(?)at different time points(day 3,7,and 28 post-surgery)during the bone regeneration process of bone defect.Conditioned medium(CM)from MOBs and FOBs were collected for culturing with BMDMs to compare the effects of MOB-CM and FOB-CM on the viability,apoptosis levels,migratory capability,phagocytic function,and cell polarization,as well as on the impact of M1/M2-BMDMs after targeted induction of differentiation.ResultsMicro-CT and histological staining results revealed that both MOBs and FOBs were able to promote the regeneration of cranial bone defects,with the superior outcomes of MOBs than FOBs.Immunohistochemical staining results showed that MOBs could significantly affect the expression of M(?)-related polarization markers and inflammation-related factors in the cranial regeneration process.On day 3 after surgery,there were significantly more i NOS-positive(M1-M(?)polarization markers)cells in the MOBs group,whereas there were few Arg1-positive(M2-M(?)polarization markers)cells,and the expression of both IL-1βand IL-10 increased significantly.By the day 7,there was a marked decrease in i NOS-positive cells number and the expression of IL-1β,whereas Arg1-positive cells number and the expression of IL-10 both increased significantly.The expression of Arg1,IL-1βand IL-10 in the MOBs group on day 28post-surgery was remained more pronounced.The results of cellular experiments showed that MOB-CM and FOB-CM had no significant effect on the viability of BMDMs,slightly inhibited apoptosis of BMDMs,apparently promoted phagocytosis,migration and NO production of BMDMs as well as inhibited osteoclastic differentiation of BMDMs.Among them,the effect of MOB-CM on BMDMs was more evident than that of FOB-CM.Both MOB-CM and FOB-CM could promote the polarization markers i NOS and Arg1 expression in BMDMs,and also increase the expression of M1-M(?)-related cytokines and IL-10,with the effect of MOB-CM was more obvious.For the M1 and M2-BMDMs,which were targeted induced polarization,MOB-CM and FOB-CM both promoted the Arg1 expression and osteogenesis-associated cytokines in M1-BMDMs and inhibited the expression of pro-inflammatory factors,which favored the conversion from M1 to M2;however,the modulation effect of them both on M2-BMDMs was very limited.Above all,the effects of MOB-CM were more significant than those of FOB-CM.ConclusionsBoth MOBs and FOBs are able to achieve cranial defects regeneration in rats,and there are site-specific effects among these interactions,with the MOBs being superior to FOBs.MOBs and FOBs can modulate the phenotype and function of BMDMs,with MOBs having a more pronounced effect,which is more conducive to the formation of an immune microenvironment for bone tissue regeneration.Part Two:Effects and mechanisms of mandible and femur osteoblasts on bone marrow-derived macrophages in the inflammatory microenvironmentObjectivePeriodontitis is a chronic infectious disease caused by periodontopathogenic bacteria.Porphyromonas gingivalis(Pg),as the main periodontal pathogen,is closely related to the development of periodontitis,and its main virulence factor is lipopolysaccharide(LPS).It has been demonstrated that Pg-LPS not only plays an important role in periodontitis-induced alveolar bone destruction,but also diffuses through the blood circulation to all organs of the body,thus affecting systemic health.In the first part of this study,we found that OBs from mandible and femur have sitespecific differences in their effects on BMDMs.In this part of the study,we intend to further investigate the variations in the effects of MOBs and FOBs on BMDMs and their mechanisms in the Pg-LPS-induced inflammation microenvironment.That will provide experimental evidence for clarifying how OBs influence the periodontitis progression via M(?) and the potential connections between periodontitis and systemic bone metabolism disorders.MethodsMOBs and FOBs were indirectly co-cultured with BMDMs under the effect of PgLPS using Transwell chambers respectively,and the roles of MOBs and FOBs on the phenotypes and functions of BMDMs and their mechanisms in the inflammatory microenvironment caused by Pg-LPS were explored by Western Blot,high-throughput whole transcriptome sequencing and RT-qPCR,etc.ResultsIn Pg-LPS-induced inflammatory microenvironment,both MOBs and FOBs were able to reduce the STAT1 phosphorylation level of BMDMs,thereby significantly inhibiting the expression of pro-inflammatory factors IL-1β and TNF-α.Meanwhile,they also promoted the expression of the M2-M(?) marker Arg1 by upregulating the STAT6 phosphorylation level.In addition,FOBs promoted the polarization of BMDMs toward M2-M(?) more intensively in effect of Pg-LPS.ConclusionsIn the presence of Pg-LPS,MOBs and FOBs were able to reduce the inflammatory response of BMDMs and induced their polarization to M2-M(?),with FOBs having a more pronounced effect.更多还原