【作者】 吴迪；

【导师】 周余来；

【作者基本信息】 吉林大学 ， 生物医学工程， 2020， 硕士

【摘要】 牙齿是人类生长发育过程中重要的器官,牙齿损伤和牙齿脱落已成为最常见的口腔疾病,牙齿缺失会导致咀嚼、语言和美观等方面出现问题,会不同程度地影响人类的生活质量。现有传统的牙齿修复方法,存在使用寿命短、使用感受差、生物相容性差等问题。因此,如何获得在功能和形态上类似天然牙齿的再生牙齿成为目前口腔医学领域研究的热点。牙齿的发育依赖于来自口腔上皮和周围的间充质之间的相互作用,同时受到各种信号通路的调控。在牙齿形成过程中,组织之间的相互作用表现为,其中一种组织产生诱导信号,另一种组织接收并响应信号,前一种组织具有的诱导能力称为牙源性潜能(odontogenic potential)。上世纪的经典重组实验证实了牙源性潜能首先出现在牙胚上皮中,继而在蕾状期(embryo day 12-13,E12-13)转移到间充质中。可见,早期的牙胚上皮可以诱导牙源性或非牙源性的间充质形成牙齿;而蕾状期之后的牙胚间充质可以诱导牙源性上皮或非牙源性上皮形成牙齿。由此,帽状期E14.5的小鼠牙胚间充质细胞常常作为构建再生牙齿的诱导信号细胞来源。然而,新鲜分离的牙胚间充质细胞数量十分有限,大大制约了再生牙齿相关的体外构建研究。本课题组此前已发现E14.5的小鼠牙胚间充质细胞在体外培养过程中易丧失牙源性潜能(MEF培养基),通过初步优化也仅可以在体外持续维持其牙源性潜能48小时(无血清KSR培养基,knockot serum replacement,KSR)。为进一步延长体外培养的牙胚间充质细胞牙源性潜能的维持时间,明确体外培养过程中牙源性潜能变化的机制,本课题中我们在无血清的培养条件基础上进一步优化了牙胚间充质细胞的体外培养体系,并通过对优化前后的体外细胞样品进行转录组测序,从而解析体外维持牙源性潜能的关键因子,为全牙再生中启动诱导成牙信号的研究提供相关的科学依据。在本研究中,我们建立了一种新型的培养体系,即无血清新型培养基(简称NM)。通过与MEF(DMEM+10%FBS)、KSR(DMEM+20%KSR)两种培养体系的对比,我们对分离的E14.5牙胚间充质细胞在三种培养体系中的生长状态进行观察,通过实时定量PCR鉴定细胞中成牙相关基因的表达,并收集不同培养条件下不同时间点的体外培养细胞,与E14.5无诱导能力的牙源性小鼠牙胚上皮重组获得重组牙胚样品,继而移植到小鼠肾包膜中进行重组样品的成牙能力鉴定。重组样品移植3周后被取出,并进行相关的成牙情况检测及组织学鉴定。此外,我们通过转录组测序分析不同培养条件下的间充质细胞样品转录组之间的差异,从而尝试解析牙源性潜能维持的机制。实验结果显示,牙胚间充质细胞在MEF和KSR培养基中贴壁生长,形态呈长梭形;在NM培养基中细胞成簇,培养4天后部分细胞悬浮成球,这说明NM促进细胞悬浮培养,可以模拟立体培养环境。我们进一步发现,牙齿发育相关基因Dlx2、Lhx6、Lhx8、Msx2、Msx1在NM组中的表达水平高于MEF和KSR组。随着传代次数的增加,NM中间充质细胞在成牙基因的表达水平呈梯度下降,特别是Msx1的表达量下调了40%,这些数据说明了NM中体外培养的间充质细胞的牙向发育的相关能力在传代过程中逐渐丧失。重组实验移植物的组织学检测结果显示,在MEF和KSR培养基中体外培养24小时的牙胚间充质细胞,分别与E14.5牙胚上皮重组后均可以形成牙齿,且成牙率为100%,但体外培养时间超过24小时的间充质细胞与E14.5天的牙胚上皮重组后无法形成牙齿。在NM中培养1、4、7天的细胞与E14.5天的牙胚上皮重组后均获得了牙齿样结构,且呈现了较高的成牙率,分别为83.3%、58.3%和66.7%,但是NM组中传代后的细胞参与构建的重组样品无法形成牙齿。转录组测序数据显示,体外培养牙胚间充质细胞在不同培养条件下及培养时间点呈现明显的基因表达差异。其中,NM条件下培养4天的细胞组(NM成牙组)和MEF条件下培养4天的细胞组(MEF不成牙组)中Bmp4的表达呈现显著差异,即Bmp4在成牙组中呈现表达水平较低,而在不成牙组中呈现表达水平较高。为了明确Bmp4在成牙能力中的作用,我们分别对NM成牙组和MEF不成牙组的培养体系里添加了BMP4蛋白和BMP通路的小分子抑制剂DM(dorsomorphin),体外培养4天后收取细胞与E14.5牙胚上皮重组并移植到肾包膜中。3周后,我们发现NM成牙组在添加了BMP4后,成牙率由原来的90%左右降为0,而MEF不成牙组在添加了DM后,成牙率由0升高为50%。这一结果说明,BMP4蛋白的添加会抑制体外牙胚间充质细胞牙源性潜能的维持,而抑制BMP通路可在一定程度上促进体外牙胚间充质细胞牙源性潜能的维持。由此可见,Bmp4在调控牙胚间充质细胞体外培养过程中成牙能力的维持上起到了重要作用。综上所述,我们优化的NM无血清培养基,能够在体外维持牙胚间充质细胞的牙源性潜能7天,并有效提高了与E14.5牙胚上皮重组的成牙率。根据转录组测序发现,优化前后的MEF培养体系和NM培养体系中的间充质细胞在基因表达上呈现显著差异,并明确了Bmp4对调控体外培养的间充质细胞的牙源性潜能的维持起到了重要作用。本研究的结果不仅为牙胚间充质细胞的体外培养优化提供了科学依据,也为构建再生牙齿提供了维持诱导成牙能力的思路。更多还原

【Abstract】 Teeth are important organs in the process of human growth and development.Tooth injury or loss have become common diseases in stomatolog y.Tooth loss could lead to prob lems in ch ewing,langu age an d aesth etics,which would affect the quality of human life to varying degrees.Traditional dental restoration method s displ ay short service life,poor sense of use,poor biocompatibility and other problems.Therefore,how to obtain the regenerative teeth similar to natural teeth in function and morphology has become a hot topic in the field of stomatology.Tooth develop s from the interactio ns b etween the oral epith elium and the surrounding mesen chyme,which i s regulated by v arious signalin g pathway s.During odonto genesis,int eractions b etween tissues are manifested by the fact that one tissue capable of producing the induction signal and the other tissue capable of receiving and respondi ng to the signal.This induction ability of the former tissue is called odontogenic potential.It is well kno wn that the o dontogenic potential shifts from dent al epithelium to dental mesenchym e at bud stage(embryo day 12-13,E12-13).Thus,the early dental epithelium can induce dental or non-d ental mesen chyme to form teeth,while the dental mesench yme after bud stage can induce dent al or non-dental epithelium to form teeth.Based on thi s,mouse d ental mesen chymal cells at cap stage(E14.5)were widely appli ed as an induction sign al cell source to toot h regeneratio n.Howev er,the number of freshly i solat ed dent al mesench ymal cells i s limited,significantl y restricting the reg eneration st udies of teeth.Previousl y,we have identified that the mouse dental mesenchymal cell s(m DMCs)lose their odontogen ic potential after 2 4 hours during the in vit ro culture wit h MEF medium.The odontogenic pot ential co uld be retained till 48 hour s in the optimization syst em with KSR medi um(knockot serum replacement).In order to further maintain the odontogenic potential of m DMCs in vitro and to clarify the und erlying changes,we here optimized the culture syst em of m DMCs in vitro and analyzed the possibl e key factors for maintaining odontogenic pot ential by transcriptome sequencing,which may provid e clues for the study of tooth regenerati on.In this study,we set up a new culture system(NM)based on previous optimized serum-free condition.Com pared with MEF medium(DMEM+10%F BS)and KSR m edium(DME M+20%KS R),we observed t he growth of cells und er microscope an d detected th e expressi on of odontogenic-rel ated gen e expression s in m DMCs in three different culture condition s.The cultured cells were further reaggregated from different media at different time points and recomb ined with E14.5 mouse dental epithelium without odonto genic potential.The recombinants were then transplant ed into adult male mouse subr enal capsule.The grafts were dissected after 3 weeks and examin ed histolo gically.Moreover,transcriptome sequen cing was used to detect differences in the transcriptome of cultured DMCs with different culture media and analyze the mechani sm of odontogeni c potential mai ntenance.As results,cultured DMCs adhered to the plates and grew with the long spindle shap e in MEF and KSR mediu m conditions.Whereas,cultured DMCs were cluster ed in NM medium.Some cells were su spend ed in pellets after 4 days of culture,which indicated that NM serum-free medium promoted cell suspen sion culture and could simulate the three-dimen sional culture environment.The expression of genes related to odontogen esi s was detected by q PCR.It was found that the expressio n level s of Dlx2,Lhx6,Lhx8,Msx2 and Msx1 in NM group was higher than those in MEF and KSR groups.As the number of passages increased,the gene expression level s in NM decreased gradu ally.Especially,the expressio n of Msx1 was detected to decrease by 40%,indicating that the odontogenic ability of DMCs in NM graduall y lost during p assage.A ccording to the result s of cell recombination tran splantati on and histologi cal examination,cell s cultured for 1 day in MEF and KSR medium were respectively recombin ed with E14.5 dental epith elium and the graft s wer e detected to form teeth with a 100% success rate,whi le the cell s cultured for more than 1 day cann ot form teeth after recombination with E14.5 dental epith elium.On the other hand,the cells cultured for 1,4,and 7 days in NM could produce teeth after recombination with E14.5 dental epit h elium with success rate 83.3%,58.3% and 66.7% respectively.But these cells failed to form teeth after passage in NM either.Transcription sequen cing data displayed the obviou s differences betw een groups in different culture system s with different culture time point s.In particularly,a great difference in the expression of Bmp4 was detected between the odontogen ic group in 4-day culture with NM and the non-odo ntogeni c group in 4-day culture with MEF,showing a lower expression lev el of Bmp4 in the odontogenic group and a high er expression level of Bmp4 in the non-odonto genic group.In order to clarify the role of Bmp4 in odontogenic potential,we suppl emented BMP4 into th e NM odontogen ic group and BMP inhibitor DM(dorsomorphin)into MEF non-odo ntogeni c group resp ectively.We f uther harvest ed teeth from the MEF non-od ontogeni c group supplyment ed with DM at a su ccess rat e up to 50%,indicating the odontogenic potenti al o f cultured cells in MEF sy stem was resued partly.Neverth eless,no teeth were found in the NM odontogenic group added with BMP4,sug gesting BMP4 may inhibit th e maintainan ce of odontogeni c potential of D MCs in vitro.Taken together,our results d emonstr at ed that optimized N M serum-free medium can maintai n the odont ogenic potenti al of DMCs for 7 days in vitro through a 3 D microen vionmen t.This optimized sy stem co uld effectively improve th e rate of tooth formation after recombinati on with E14.5 dental epithelium.According to transcriptom e sequencing data,great differences of odontgenic relat ed gen e expression s were detected in the cutured cells before and after optimization.One of the differenti ally expressed genes,Bmp4,was selected for validation.And it was found that Bmp4 plays a key role i n the formation of tooth,and BMP4 inhibitor D M can restore th e odontog enic abilit y of cult ured cells.These result s may provide a new sight for the culture of DMCs in vitro and a possible way for regulation of activating signal s in the constr uction of regenerativ e teeth.更多还原