【作者】 刘畅；

【导师】 彭彬；

【作者基本信息】 武汉大学 ， 牙体牙髓病学， 2023， 博士

【摘要】 第一部分BACH1在人健康牙髓组织的表达和分布研究目的:研究BTB和CNC同源物1(BTB and CNC homology 1,BACH1)在人健康牙髓的表达及分布情况。研究方法:收集符合纳入标准的人健康的磨牙或前磨牙。蛋白免疫印迹法(Western blot analysis,WB)检测BACH1在人健康牙髓组织的表达。经脱钙及切片后进行免疫组化染色和组织免疫荧光染色检测BACH1在人健康牙髓的分布特点。实验结果:WB结果显示人健康牙髓组织表达BACH1。染色结果显示BACH1在人健康的牙髓组织呈阳性表达,且相较多细胞层其在成牙本质细胞层的表达显著增强。结论:BACH1在人健康的牙髓组织广泛分布且在成牙本质细胞层高表达,提示BACH1可能参与牙本质的分泌和牙髓牙本质复合体稳态的维护。第二部分BACH1在人牙髓干细胞(human dental pulp stem cells,h DPSCs)体外成牙本质向分化过程中的表达变化和调控方式研究目的:探索h DPSCs体外成牙本质向分化过程中BACH1的表达变化及其相关调控因素。研究方法:体外分离培养原代细胞,利用细胞免疫荧光染色、流式细胞术和多向诱导分化的方法明确细胞类型为h DPSCs。体外诱导h DPSCs成牙本质向分化,通过碱性磷酸酶(alkaline phosphatase,ALP)染色和ALP活力检测,成牙本质向分化的相关标志物runt相关转录因子2(runt-related transcription factor 2,RUNX2)、牙本质基质蛋白1(dentin matrix acidic phosphoprotein 1,DMP1)和牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)表达水平的检测,以及茜素红S(alizarin red S,ARS)染色来确认h DPSCs发生了成牙本质向分化。通过WB和实时定量聚合酶链式反应(real-time quantitative polymerase chain reaction,RT-q PCR)检测BACH1总蛋白、胞核蛋白和信使核糖核酸(messenger RNA,m RNA)水平的表达。细胞免疫荧光染色观察h DPSCs成牙本质向分化过程中BACH1在h DPSCs分布的变化。使用特异性抑制剂对BACH1的调控方式进行探索。实验结果:在体外诱导h DPSCs成牙本质向分化的过程中,RT-q PCR结果显示BACH1的表达在转录水平没有发生显著变化。WB结果显示在分化早期BACH1总蛋白下降,随后逐渐恢复。对于胞核BACH1细胞免疫荧光和WB结果显示在成牙本质向诱导分化的早期就出现明显下降,随后逐渐增加,尽管第14天的BACH1核蛋白水平仍略低于第0天。使用丝裂原活化蛋白激酶(p38 mitogen-activated protein kinases,p38 MAPK)抑制剂SB203580和染色体维持蛋白1(chromosomal maintenance 1,CRM1)抑制剂来普霉素B(leptomycin B,LMB)均可抑制BACH1的核输出。与此同时,使用SB203580可以抑制在矿化诱导液刺激下胞浆CRM1水平的降低。使用蛋白酶体抑制剂MG132(ZLeu-Leu-Leu-al)则可以抑制BACH1总蛋白的降低。结论:在h DPSCs体外成牙本质向分化过程中,p38 MAPK/CRM1轴介导BACH1的核输出,其亚细胞分布改变并且通过蛋白酶体途径发生降解。第三部分BACH1在h DPSCs增殖、迁移及成牙本质向分化中的作用的体外研究研究目的:研究BACH1表达下调对h DPSCs增殖、迁移和成牙本质向分化的影响。研究方法:利用慢病毒载体对h DPSCs进行转染,构建BACH1低表达的h DPSCs(LVsh BACH1)。采用RT-q PCR、WB和观察绿色细胞荧光(green fluorescent protein,GFP)的方法来验证病毒转染的效率。利用CCK-8(cell counting kit-8)和5-乙炔基-2’-脱氧尿苷(5-Ethynyl-2’-deoxyuridine,Ed U)法检测细胞增殖活力。碘化丙啶(propidium iodide,PI)染色检测细胞周期。划痕实验和Transwell实验测定细胞迁移能力。在成牙本质向诱导后第7天进行ALP染色和ALP活性测定;第14天时检测成牙本质向相关标志物RUNX2、DMP1和DSPP的m RNA和蛋白质表达水平;第21天时进行ARS染色和吸光度测定用以评估h DPSCs成牙本质向分化的情况。实验结果:与对照组相比,CCK-8结果显示LV-sh BACH1组增殖减弱;Ed U法染色结果显示LV-sh BACH1组Ed U阳性细胞比例降低;PI染色结果显示LV-sh BACH1组处于合成期(synthesis phase,S phase),DNA合成后期(gap 2 phase,G2 phase)/有丝分裂期(mitotic segregation phase,M phase)的细胞比例较低,而在静止期(cell quiescence phase,G0 phase)/DNA合成前期(gap 1 phase,G1 phase)比例较高。划痕实验结果表明LV-sh BACH1组伤口愈合百分比从12小时起就出现差异,较对照组减少,划痕宽度在24小时时明显增宽;Transwell实验结果显示LV-sh BACH1组穿过小室的细胞数量有所减少。在成牙本质向分化过程中,LV-sh BACH1组在第7天表现出ALP染色颜色减弱和ALP活性降低,第14天RUNX2、DMP1和DSPP表达下调,第21天钙结节形成减少。结论:在h DPSCs内下调BACH1的表达可引起细胞增殖抑制,细胞周期停滞,迁移能力降低,成牙本质向分化的潜力减弱,表明BACH1是h DPSCs增殖、迁移和成牙本质细胞分化的重要调节因子。第四部分BACH1调控h DPSCs成牙本质向分化的相关机制研究研究目的:探索BACH1低表达引起h DPSCs成牙本质向分化抑制的潜在机制。实验方法:分离培养来自三个不同个体的h DPSCs,利用慢病毒载体对h DPSCs进行转染,构建BACH1低表达组(LV-sh BACH1)和含空载体的对照组(LV-NC),然后进行成牙本质向分化的诱导。诱导3天后,提取样本内总RNA并进行相关的样本处理和RNA高通量测序(RNA-sequencing,RNA-seq),根据相应的入选标准筛选出差异基因。利用GO数据库和MSig DB数据库进行生物信息学分析,在前期研究的基础上结合生物信息学分析的结果挖掘在BACH1在成牙本质向分化过程中所涉及的可能的信号通路,并采用特异性的抑制剂或激活剂进行验证。为探究相关通路的作用,利用相关通路的激活剂或者抑制剂干预h DPSCs的成牙本质向分化诱导,并对成牙本质向分化相关的指标进行检测,采用的方法与之前相同。实验结果:LV-sh BACH1组血红素加氧酶1(heme oxygenase-1,HO-1)表达上调。加入HO-1抑制剂锡原卟啉IX(tin-protoporphyrin IX,Sn PP),在LV-sh BACH1+Sn PP组中,所有成牙本质向分化的相关指标检测结果均显示减弱。生物信息学分析结果显示Wnt/β-Catenin信号通路明显富集,富集分数峰值在下方,基因集在底部富集,该通路在LVsh BACH1组受抑制。所有基因的启动子区域和转录因子结合位点的富集结果包含淋巴增强结合因子1(lymphoid enhancer binding factor 1,LEF1)。矿化诱导1天后,细胞免疫荧光显示LV-sh BACH1组β-Catenin入核受阻;矿化诱导3天后,RT-q PCR的结果表明LV-sh BACH1组部分Wnt/β-Catenin相关基因m RNA表达降低;WB结果显示β-Catenin总蛋白水平和胞核蛋白水平降低,激活的β-Catenin减少。加入Wnt/β-Catenin信号通路激活剂氯化锂(lithium chloride,Li Cl),LV-sh BACH1+Li Cl组在第7天ALP活力部分恢复。随后,成牙本质向分化的相关因子m RNA水平以及ARS染色的结果也表现出了相同的趋势。结论:BACH1调控h DPSCs成牙本质向分化并非HO-1依赖而是有赖于Wnt/β-Catenin信号通路。BACH1部分通过调节Wnt/β-Catenin信号通路在h DPSCs的成牙本质向分化过程中发挥作用。这些发现为BACH1在h DPSCs成牙本质向分化中的作用提供了新的思路,但还需要进一步的研究来充分阐释BACH1对h DPSCs再生能力的影响。更多还原

【Abstract】 Part Ⅰ Expression and distribution of BACH1 in human healthy dental pulp tissues Aim: To explore the expression and distribution of BACH1 in human healthy dental pulp tissues.Methods: Healthy human molars or premolars that met the inclusion criteria were collected.WB was conducted to detect the expression of BACH1 in human healthy dental pulp tissues.Immunohistochemical staining and tissue immunofluorescence staining were used after decalcification and sectioning to detect the distribution characteristics of BACH1 in human healthy dental pulp tissues.Results: The results of WB showed that BACH1 was expressed in human healthy dental pulp tissues.The staining results demonstrated that BACH1 was positively expressed in human healthy pulp tissues,and the expression was significantly enhanced in the odontoblastic layer than in the cell rich zone.Conclusion: BACH1 is widely distributed in healthy human dental pulp tissues and highly expressed in the odontoblast layer,suggesting that BACH1 may be involved in the dentin secretion and maintenance of dentin-pulp complex homeostasis.Part Ⅱ Expression patterns and upstream regulation of BACH1 during the odontoblastic differentiation of human dental pulp stem cells(h DPSCs)in vitroAim: To investigate the modulation and regulators of BACH1 expression during the odontoblastic differentiation of h DPSCs in vitro.Methods: Primary cells were isolated and cultured,and the cell type was identified as h DPSCs using cell immunofluorescence staining,flow cytometry and multi-directional induction of differentiation.The odonto/osteogenic differentiation of h DPSCs was induced in vitro,and was confirmed by detecting ALP activity,expression of mineralization markers RUNX2,DMP1 and DSPP,and calcium deposit.The expression levels of total BACH1 protein,nuclear protein and m RNA were measured by WB and RT-q PCR.Cellular immunofluorescence staining was performed to observe the change of subcellular distribution of BACH1 in h DPSCs during the differentiation process.The mode of regulation of post-translational modification of BACH1 was explored by specific inhibitors.Results: The odontoblastic differentiation of h DPSCs was induced in vitro,during which the total BACH1 protein decreased initially and then recovered gradually,while m RNA levels did not change significantly.Cellular immunofluorescence and WB results showed a significant downregulation in BACH1 nucleoprotein in the early stage of the differentiation process,followed by a gradual increase,although BACH1 nuclear protein levels were still slightly lower on day 14 than on day 0.The nuclear export of BACH1 was inhibited by the p38 MAPK inhibitor SB203580 and CRM1 inhibitor LMB.Meanwhile,the use of SB203580 inhibited the reduction of cytoplasmic CRM1 in response to odonto/osteogenic induction medium(OM)stimulation.The use of MG132,inhibited the reduction of total BACH1 protein.Conclusion: The decreased level of BACH1 total protein is due to proteasomal degradation and nuclear export of BACH1 is mediated by p38 MAPK/CRM1 axis during the differentiation of h DPSCs into odontoblastic-like cells in vitro.Part Ⅲ Effects of BACH1 on the proliferation,migration and odontoblastic differentiation of h DPSCs in vitroAims: To study the role of BACH1 downregulation in the proliferation,migration and odontoblastic differentiation of h DPSCs.Methods: The h DPSCs were transfected with a lentiviral vector to construct BACH1-knockdown h DPSCs(LV-sh BACH1 group).RT-q PCR,WB and observation of GFP were used to verify the knockdown efficiency.Cell proliferation viability was detected using CCK-8 and Ed U assay.The cell cycle was discerned by PI staining.Cell migration ability was determined by scratch assay and Transwell assay.ALP staining and activity were performed on day 7 after OM treatment;m RNA and protein expression of odontoblastic-related factors RUNX2,DMP1 and DSPP were measured on day 14;ARS staining assay was conducted on day 21 to assess the degree of the odontoblastic differentiation of h DPSCs.Results: The results of CCK-8 displayed that the proliferation viability of the LV-sh BACH1 group was decreased,and the percentage of Ed U-positive cells in the LV-sh BACH1 group was lower than that in the control group.The BACH1-knockdown group showed a lower proportion of S and G2/M phases and a higher proportion of G0/G1 phases.The percentage of closed wound area in the LV-sh BACH1 group was lower than that in the control group from 12 hours,and the wound width was significantly wider at 24 hours.In the LV-sh BACH1 group,ALP staining and ALP activity were decreased on day 7,RUNX2,DMP1 and DSPP expression was attenuated on day 14,and calcium nodule formation was reduced on day 21.Conclusion: Downregulation of BACH1 inhibits cell proliferation,induces cell cycle arrest,weakens migration and reduces the odontoblastic differentiation potential of h DPSCs,suggesting that BACH1 is an important regulator of the proliferation,migration and odontoblastic differentiation of h DPSCs in vitro.Part Ⅳ The molecular mechanism by which BACH1 functions in the odontoblastic differentiation of h DPSCsAims: To explore the potential mechanism of inhibition of odontoblastic differentiation of h DPSCs induced by BACH1 knockdown.Methods: h DPSCs from three different individuals were stably transfected with lentivirus to construct BACH1-knockdown h DPSCs(LV-sh BACH1 group)and a control group containing an empty vector(LV-NC)for OM induction.After 3 days of induction,total RNA was extracted from the samples and RNA-seq was performed.Screen out the different expression genes according to the corresponding filter criteria.Based on the results of previous studies and bioinformatics analysis,the possible signaling pathways involved in the differentiation of BACH1 into odontoblastic-like cells were excavated and then validated by a specific inhibitor or activator.To investigate the role of related pathways,the activator or inhibitor of related pathways was administrated.The methods of monitoring the extent of odontoblastic differentiation are the same as before.Result: As the expression of HO-1 was increased in the LV-sh BACH1 group,the HO-1inhibitor Sn PP was added.In the LV-sh BACH1+Sn PP group,all tests related to odontoblastic differentiation showed diminished results.Bioinformatics analysis showed significant enrichment of the Wnt/β-Catenin signaling pathway with the peak of enrichment score at the bottom and the gene set was enriched below,which means the gene set was inhibited in the LVsh BACH1 group.Enrichment results for promoter regions and transcription factor binding sites of all genes contained LEF1.After 1 day of mineralization induction,cellular immunofluorescence displayed that β-Catenin nuclear import was reduced in the LV-sh BACH1 group.After 3 days of mineralization induction,results by WB and RT-q PCR showed that the m RNA levels of some Wnt/β-Catenin signaling pathway related genes were reduced in the LVsh BACH1 group,and total protein levels,nuclear protein level and activation level of β-Catenin were decreased as well.With the addition of Li Cl,a Wnt/β-Catenin signaling pathway activator,the LV-sh BACH1+ Li Cl group demonstrated partial restoration of ALP activity on day 7.Subsequent m RNA levels of mineralization markers and the results of ARS staining assay displayed the same trend.Conclusion: BACH1 regulates odontoblastic differentiation of h DPSCs not dependent on HO-1 but related to the Wnt/β-Catenin signaling pathway.These findings shed new light on the function of BACH1 in the odontoblastic differentiation of h DPSCs,but additional research is required to fully characterize the role of BACH1 in the regenerative capacity of h DPSCs.更多还原