【作者】 马莉；

【导师】 曹正国；

【作者基本信息】 武汉大学 ， 口腔临床医学， 2022， 博士

【摘要】 第一部分:小鼠根尖周炎模型中P.gingivalis对成牙骨质细胞分化和自噬的影响目的牙龈卟啉单胞菌(Porphyromonas gingivalis)与牙周炎和根尖周炎均密切相关。本部分旨在阐明小鼠根尖周炎(apical periodontitis,AP)模型中,沿根管系统浸润根尖组织的P.gingivalis对根尖周成牙骨质细胞分化及自噬的影响。方法小鼠麻醉后,用高速手机打开左下颌第一磨牙髓腔,保持髓腔开放建立AP模型,以模拟体内成牙骨质细胞的炎症状态。其中一半开髓小鼠接受重复细菌处理(一周三次),即用2%CMC混悬P.gingivalis,混悬液通过口腔注射进入开放的髓腔。保留未经细菌处理小鼠的右下颌第一磨牙作空白对照。3周后取材,使用X线排除髓室顶未充分揭开或髓室底穿孔的组织标本,并初步判断根尖区域骨吸收程度。通过micro-CT检测三组(健康组、AP组、AP+P.gingivalis组)第一磨牙根尖区组织破坏情况。q PCR检测根尖周组织中Il-6和Tnf-α的表达水平。采用H&E染色判断该AP模型中的炎症细胞浸润情况。通过组织免疫荧光验证P.gingivalis可否通过根管系统进入根尖区。使用免疫组织化学检测根尖部成牙骨质细胞及附近牙周膜细胞中成骨标志物Osterix、自噬标志物LC3及p62的表达情况。结果1.X线和micro-CT结果显示,髓腔开放法可成功建立小鼠AP模型,开髓组第一磨牙根尖区可见明显骨质破坏,且三组(健康组、AP组、AP+P.gingivalis组)组织破坏范围逐渐增大。2.与健康对照组相比,AP组根尖周组织中Il-6和Tnf-α的表达明显升高。H&E染色显示AP组、AP+P.gingivalis组第一磨牙根尖区均可见明显炎症细胞浸润。免疫荧光提示髓腔开放合并口腔注射P.gingivalis可使细菌到达根尖区,进而与成牙骨质细胞相互作用。3.免疫组化结果证实,三组(健康组、AP组、AP+P.gingivalis组)第一磨牙根尖区成牙骨质细胞中成骨标志物Osterix的表达依次降低。相较于AP组,AP+P.gingivalis组第一磨牙根尖区成牙骨质细胞中自噬标志物LC3表达增多,而自噬底物p62表达降低,这也是首次在体内模型中检测成牙骨质细胞自噬活动。结论P.gingivalis加重小鼠AP模型中的根尖周组织破坏情况,并可抑制根尖周成牙骨质细胞分化以及激活细胞自噬活动。但是,P.gingivalis如何调控成牙骨质细胞分化矿化及自噬活动,其具体机制尚需进一步探索。第二部分:CXXC5通过MAPK信号网络调控P.gingivalis抑制的成牙骨质细胞分化矿化目的CXXC型锌指蛋白5(CXXC-type zinc finger protein 5,CXXC5)是一种具有调控基因表达和信号传导功能的转录因子。本部分旨在探究P.gingivalis抑制成牙骨质细胞分化过程中CXXC5所起的作用,阐明CXXC5调控成牙骨质细胞分化矿化的潜在机制。方法在体外,对小鼠成牙骨质细胞系OCCM-30进行不同时间及不同浓度的P.gingivalis处理,通过检测促炎细胞因子Il-6、Mcp-1和Rantes的表达,确定体外细胞炎症模型的建立。在该模型中检测成骨标志分子Osterix、OCN和BSP的表达以及矿化结节的形成。对OCCM-30矿化样本进行转录组测序,并通过q PCR验证细胞矿化过程中成骨标志物及Cxxc5的表达。免疫荧光显示P.gingivalis作用下CXXC5的胞内分布及表达情况。在体内,建立小鼠AP模型并加入P.gingivalis刺激,q PCR及免疫组化检测根尖区CXXC5的表达情况。此外,构建CXXC5的siRNA和过表达质粒并分别转染到OCCM-30中,通过q PCR和Western blotting检测CXXC5表达调控后成骨相关基因和蛋白的表达水平,通过ALP染色及活性实验检测细胞矿化情况。Western blotting检测CXXC5敲低或过表达后多种信号通路的活性变化,并使用特异性抑制剂阻滞MAPKs和PI3K-Akt信号通路,以证实它们直接参与调控CXXC5促进OCCM-30分化矿化的过程。结果1.P.gingivalis可呈浓度依赖性诱导OCCM-30细胞产生促炎因子,并减弱细胞的矿化能力。2.CXXC5在OCCM-30细胞矿化过程中逐渐积累,而在P.gingivalis处理的细胞和AP模型中表达明显减少。3.用siRNA干扰CXXC5显著抑制OCCM-30细胞分化,具体表现为成骨标志物Osterix、OCN和ALP活性的下降。反之,CXXC5过表达可促进细胞分化,从而部分减弱P.gingivalis对OCCM-30成骨分化的抑制作用。4.Erk1/2、p38和PI3K-Akt在CXXC5干扰时被抑制,在CXXC5过表达时被激活,而Wnt/β-catenin呈现相反变化趋势。使用p38、PI3K-Akt特异性抑制剂可减弱CXXC5对OCCM-30细胞分化的促进作用,而抑制Erk1/2反而加强这一作用。结论P.gingivalis刺激抑制成牙骨质细胞分化矿化能力,同时降低CXXC5表达水平,而过表达CXXC5可部分减弱P.gingivalis对细胞分化的抑制效应。本部分研究表明,CXXC5过表达可能有利于P.gingivalis侵犯下受损牙骨质的再生,并提示MAPK信号网络在此过程中具有重要的调控作用。CXXC5是牙骨质重建的潜在治疗靶点,有望促进牙骨质再生。第三部分:CXXC5协同Stat3/Erk/Akt信号网络调控P.gingivalis诱导的成牙骨质细胞自噬活动目的P.gingivalis可在多种牙周组织细胞中诱导炎症及自噬活动,而CXXC5的表达在P.gingivalis刺激时显著下调。本部分旨在成牙骨质细胞中检测P.gingivalis相关自噬活动,探究CXXC5在P.gingivalis诱导的OCCM-30细胞自噬中的作用,阐明CXXC5调控成牙骨质细胞自噬活动的分子机制。方法体外建立OCCM-P.gingivalis共培养系统,检测促炎因子Il-6、Mcp-1和Rantes的水平,同时检测Jak/Stat3信号通路、自噬标志分子LC3和p62的表达。细胞免疫荧光验证P.gingivalis可否内化于OCCM-30,并观察P.gingivalis作用下LC3的胞内分布及表达情况。TEM观察P.gingivalis刺激下成牙骨质细胞内自噬相关囊泡的积累。P.gingivalis作用下检测CXXC5的m RNA和蛋白表达水平。通过在OCCM-30细胞中抑制或过表达CXXC5,探索其对促炎因子产生及自噬标志物表达的调控作用,并检测NF-κB、Jak/Stat3、PI3K-Akt和Erk1/2的变化,筛选P.gingivalis及CXXC5的下游信号通路。使用特异性抑制剂阻断Jak/Stat3、PI3K-Akt和Erk1/2后检测促炎因子及自噬标志物的变化。使用siRNA分别沉默自噬分子ATG5和Beclin1研究自噬对炎症的反馈调节作用。采用Ed U染色和Annexin V/PI试剂盒分别检测P.gingivalis刺激下的细胞增殖与凋亡。结果1.P.gingivalis可诱导OCCM-30细胞炎症与自噬活动,表现为促炎因子Il-6、Mcp-1和Rantes表达增多,Jak/Stat3信号通路激活,LC3-II积累而p62降解。细胞免疫荧光显示P.gingivalis可内化于OCCM-30细胞,使LC3荧光信号增强并聚集在细胞核周围。TEM观察到P.gingivalis刺激后胞内自噬相关囊泡结构明显增多。2.短时间P.gingivalis处理同样使CXXC5表达降低。敲低CXXC5可抑制细胞促炎因子产生与自噬活动,一定程度上限制P.gingivalis对炎症与自噬的过度激活;而CXXC5过表达可增加Il-6、Mcp-1和Rantes表达,激活Jak/Stat3信号通路,积累LC3-II并降解p62,表现为对炎症与自噬的促进作用。3.P.gingivalis刺激和CXXC5过表达都可以激活Jak/Stat3、PI3K-Akt和Erk1/2信号通路。加入相应抑制剂Stattic、LY294002和PD98059后,胞内促炎因子产生减少,自噬水平被显著抑制。此外,P.gingivalis激活自噬活动依赖于ATG5及Beclin1的活化,加入siRNA分别抑制ATG5和Beclin1后,不仅自噬活动受到抑制,胞内促炎因子的产生也明显减少。4.短时间P.gingivalis刺激可促使OCCM-30细胞中Annexin V(+)比例增加,而Ed U(+)比例明显降低,表现为凋亡促进和增殖抑制效应。此外,P.gingivalis处理可使更多的细胞产生ROS,并提高细胞平均ROS水平。结论P.gingivalis-OCCM共培养可激活细胞自噬活动并抑制CXXC5表达,而CXXC5表达降低可转而限制过度激活的自噬水平。这个过程部分依赖于Jak/Stat3、PI3K-Akt和Erk1/2的信号转导以及自噬分子ATG5和Beclin1的活化。此外,P.gingivalis可能通过维持胞内高水平自噬而影响细胞增殖与凋亡。因此,P.gingivalis可能通过炎症激活与自噬诱导两种途径对成牙骨质细胞造成损害。更多还原

【Abstract】 Part.Ⅰ: Effects of P.gingivalis on Cementoblast Differentiation and Autophagy in Apical Periodontitis Models Objective Porphyromonas gingivalis associates tightly with periodontal diseases,and it is also a dominant pathogen of periapical periodontitis.This part aims to elucidate the effects of downward-infiltrating P.gingivalis on differentiation and autophagy of tooth root surface lining cementoblasts in murine apical periodontitis(AP)models.Methods The mice were anesthetized to open the pulp chambers of the left mandibular first molars with a high-speed dental handpiece.The pulps were exposed in the oral cavity for 21 d to create an inflammatory environment for cementoblasts and establish AP models in vivo.Half of the mice were further treated with a repeated oral application of P.gingivalis(MWF/week)in a 2% carboxymethylcellulose vehicle.No surgical interventions were done on the right first molars of the mice without P.gingivalis treatment,which was maintained as the healthy control group.After animal sacrifice,X-ray photography was performed first to rule out samples with insufficient unfolding of pulp chamber roof or perforation of pulp chamber floor,and to preliminarily assess the extent of apical lesions.To clearly evaluate ranges of periapical bone destruction,the mandibles from three groups(healthy,AP,and AP plus P.gingivalis)were scanned by micro-CT.The expression levels of Il-6 and Tnf-α in periapical tissues were detected by q PCR.Inflammatory cell infiltration in apical tissues was revealed by H&E staining,and immunohistofluorescence was carried out to verify the apical invasion of P.gingivalis through root canals.Immunohistochemistry was performed to detect the expressions of osteogenic marker Osterix as well as autophagy markers LC3 and p62 in apical cementoblasts and ambient periodontal ligament cells.Results1.The murine AP model was successfully established by pulp exposure.In contrast to mandibles in the healthy group,samples in the AP group exhibited significant apical bone destruction around the first molars,and additional P.gingivalis treatment enlarged the ranges of apical bone resorption,revealed by X-ray photography and micro-CT analysis.2.The expression levels of Il-6 and Tnf-α in apical tissues of the AP group were much higher than those in the healthy group.H&E staining showed evident inflammatory cell infiltration in the AP and AP plus P.gingivalis group.Immunohistofluorescence testified that pulp exposure combined with oral application of P.gingivalis suspension enabled the bacteria to invade apical areas and further interact with tooth root lining cementoblasts.3.Immunohistochemistry results demonstrated that Osterix,LC3,and p62 were all positive in apical cementoblasts and ambient periodontal ligament cells around mandibular first molars.A sharply reduced expression of Osterix in cementoblasts was observed in the following three groups: the healthy group,the AP group,and the AP plus P.gingivalis group.Compared to the AP group,darker LC3 B staining and lighter p62 staining were observed in cementoblasts of the P.gingivalis treated AP group.This is also the first time to detect autophagy of cementoblasts in vivo.Conclusion P.gingivalis aggravated AP in mouse models,with repression of cell differentiation and activation of autophagy activities in cementoblasts.However,the molecular mechanisms concerning P.gingivalis-suppressed differentiation and P.gingivaliselicited autophagy in cementoblasts still need to be elucidated by further exploration.Part.Ⅱ: CXXC5 Mediates P.gingivalis-suppressed Cementoblast Functions Partially via MAPK Signaling Network Objective CXXC-type zinc finger protein 5(CXXC5)is a novel transcription regulator of gene expression and cell signaling.This part aims to explore the possible involvement of CXXC5 in P.gingivalis-suppressed cementoblast differentiation,and to uncover the mechanism by which CXXC5 mediates cementoblast differentiation.Methods In vitro,OCCM-30,an immortalized mouse cementoblast cell line,was treated with P.gingivalis for different time lengths or with different multiplicity of infection,and the samples were collected for detection of proinflammatory cytokines Il-6,Mcp-1,and Rantes,osteogenic markers Osterix,OCN,and BSP,as well as mineralized nodule formation.The expressions of osteogenic markers and Cxxc5 during cementoblast differentiation were analyzed by RNA-seq and testified by q PCR.Immunofluorescence was carried out to verify the intercellular distribution and expression of CXX5 upon P.gingivalis treatment.In vivo,mouse AP models with P.gingivalis invasion or not were used to examine CXXC5 expression in apical areas.Further,OCCM-30 was transfected with siRNA and overexpression plasmids of CXXC5,respectively.The knockdown,overexpression,and rescue effects were revealed in terms of associated osteogenic gene and protein expressions,as well as activities of several signaling pathways,analyzed by either q PCR or western blot analysis.Meanwhile,ALP staining and ALP activity were used to clearly determine cell mineralization.Specific inhibitors for MAPKs and PI3K-Akt signaling were employed to confirm the participation of these pathways in CXXC5-promoted cementoblast differentiation.Results1.P.gingivalis repressed the mineralization capacity of cementoblasts and induced proinflammatory cytokine production in a dose-dependent manner.2.The expression of CXXC5 decreased in P.gingivalis-treated OCCM-30 cells and murine AP models,but gradually increased during cementoblast differentiation.3.RNA interference of CXXC5 significantly inhibited cementoblast differentiation,represented by the decline of bone-associated markers Osterix,OCN,and ALP activity.CXXC5 overexpression facilitated cell differentiation,and therefore attenuated the P.gingivalis-repressed effects on cementoblast differentiation.4.Erk1/2,p38,and PI3K-Akt were inactivated by silencing CXXC5 and activated upon CXXC5 overexpression,whereas Wnt/β-catenin exhibited an opposite trend.The employment of specific inhibitors revealed that the CXXC5-dependent promotion of cementoblast differentiation was partially abrogated by p38 and PI3 KAkt inhibitors but was exacerbated by inhibiting Erk1/2.Conclusion Our data clarified that cementoblast differentiation and CXXC5 expression were repressed by P.gingivalis stimulation,and that CXXC5 overexpression could partially attenuate P.gingivalis-repressed osteogenic effects of cementoblasts.This research indicated overexpressing CXXC5 may be conducive in the regeneration of impaired cementum caused by P.gingivalis invasion and suggested that MAPK signaling network balanced the facilitation effects of CXXC5 on cementoblast differentiation.Therefore,CXXC5 is a novel potential therapeutic target for cementum reconstruction.Part.Ⅲ: CXXC5 Orchestrates Stat3/Erk/Akt Signaling Networks toModulate P.gingivalis-elicited Autophagy in Cementoblasts Objective P.gingivalis is reported to induce inflammation and autophagy in various types of periodontal cells.The expression of transcription factor CXXC5 is significantly repressed by P.gingivalis invasion.This study aims to investigate the autophagyinducing effects of P.gingivalis,the contribution of CXXC5 in autophagy regulation,and the possible involvement of signals in cementoblasts,root surface cells pivotal in the apical areas.Methods Short-term OCCM-P.gingivalis coculture system was established in vitro.The changes in levels of proinflammatory cytokines,phosphorylated Stat3,autophagy-related LC3,and p62 were examined.Immunofluorescence staining was performed to display the internalized P.gingivalis and the distribution of LC3 signals in cementoblasts.TEM was used to observe autophagic vesicle accumulation in the P.gingivalis-stimulated samples.The downregulation of CXXC5 in cementoblasts co-cultured with P.gingivalis was validated by biochemical assays.Next,cementoblasts with loss and gain of CXXC5 were developed to investigate its effect on inflammation and autophagy modulation,and changes of potentially related signaling pathways including P65,Jak/Stat3,PI3K-Akt,and Erk1/2 were evaluated.The specific inhibitors for Jak/Stat3,PI3K-Akt,and Erk1/2 were applied,and downstream proinflammatory cytokine production and autophagy-related protein expressions were determined.RNA interference of ATG5 and Beclin1 was carried out to explore the bidirectional regulation of autophagy on inflammation.Ultimate cell fate caused by P.gingivalis invasion was determined by Ed U assay and Annexin V/PI kit.Results1.P.gingivalis elicited inflammation and autophagy in cementoblasts,represented by enhanced proinflammatory chemokine production,Stat3 phosphorylation,LC3-II accumulation,and p62 degradation.Fluorescence microscopy detected bright LC3 spots around the nucleus and internalized bacteria in the P.gingivalis-infected samples.Moreover,TEM captured evident autophagic vesicles(autophagosomes and autolysosomes)in the P.gingivalis-cocultured group.2.Short-term P.gingivalis treatment decreased CXXC5 expression.The knockdown of CXXC5 attenuated the inflammatory responses and yielded an autophagy-silent effect;thus partially restricting the excessive inflammation and autophagy induced by P.gingivalis invasion.In contrast,CXXC5 overexpression elevated the levels of inflammatory factors Il-6,Mcp-1,and Rantes,activated p-Stat3 and LC3-II,and decreased p62 expression.3.Biochemical assays detected Stat3/Akt/Erk activation during P.gingivalis treatment and CXXC5 overexpression,and the employment of their inhibitors Stattic,LY294002,and PD98059 confirmed their positive influence on autophagy and inflammation.Furthermore,P.gingivalis and CXXC5 induced autophagy through ATG5 and Beclin1 activation,given specific siRNA for them significantly suppressed LC3 conversion and p62 degradation.4.In the P.gingivalis-treated cells,the percentage of Annexin V(+)cells steadily increased while the red Ed U fluorescent signals were evidently quenched.Furthermore,ROS production was triggered by P.gingivalis infection as more OCCM-30 cells produced ROS and the total amount of ROS generation was significantly enhanced.Conclusion P.gingivalis increased autophagy activities and decreased transcription factor CXXC5 expression in cementoblasts,while CXXC5 downregulation limited the extent of excessive autophagy in turn.The autophagy-inducing effects were partially dependent on Stat3/Akt/Erk signaling and autophagy-related Beclin1 and ATG5.Furthermore,P.gingivalis was likely to affect cell proliferation and apoptosis via maintenance of high autophagic levels.Therefore,P.gingivalis can cause damage to cementoblasts not only by traditional inflammation activation,but also by autophagy induction.更多还原