【作者】 张娟；

【导师】 孙宏晨；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2015， 博士

【摘要】 对于深龋和意外穿髓等患者的治疗,如何保存其患牙的牙髓活力是口腔科医生面临的重要挑战。组织学上,牙体组织由3种硬组织釉质、牙本质、牙骨质和1种软组织牙髓构成,其中,牙本质作为牙体组织主要成分,在保护牙髓、支撑釉质起重要作用。龋病、外伤、磨耗等引起的物理学、化学和生物学作用通过牙本质小管和成牙本质细胞突起而损伤牙髓,轻则引起牙髓的反应性变化、重则引起牙髓的感染和坏死。其中,成牙本质细胞形成第三期牙本质是牙髓的主要防御反应,通过形成第三期牙本质来保护牙髓免受进一步损伤。因此,如何促进损伤牙髓形成第三期牙本质是防止病变进一步发展和保存患牙活力重要的科学问题。第三期牙本质形成的过程包括牙髓干细胞的迁移、增殖和向成牙本质细胞的分化,并分泌牙本质基质。其中牙髓干细胞向成牙本质细胞的分化是修复性牙本质形成中的重要过程,多种因素、细胞因子和信号通路参与和调控了这个过程。生长因子通过促进成牙本质细胞增殖、分化和功能,进而促进第三期牙本质的形成。研究表明,受伤的牙髓细胞分泌血管内皮生长因子(VEGF)刺激血管生成,通过提高Runx2(Runt-related transcription factor 2)蛋白水平,引起牙本质涎磷蛋白(DSPP)表达等机制促进成牙本质细胞的增殖和分化,进而促进修复性牙本质形成。VEGF是一个强有力的促进血管生成的细胞因子,具有内皮细胞特异性有丝分裂原活性,在体内能够刺激血管的发生,增加血管的渗透性。因此,应用VEGF有可能通过促进成牙本质细胞增殖和分化、形成第三期牙本质,以保护牙髓。骨形成蛋白2(BMP2),属于TGF-β家族的蛋白质,是一个自分泌蛋白。BMP2具有通过Smad 1/5、p38a MAPK等信号通路促进成牙本质细胞分化和修复性牙本质的形成的作用。成牙本质细胞能产生和分泌BMP2,直接影响分泌和刺激邻近的成牙本质细胞增殖和分化导致牙本质形成。有研究通过促进BMP2蛋白表达或应用外源性BMP2蛋白实现形成修复性牙本质或第三期牙本质。而BMP2蛋白质昂贵,如果应用表达BMP2蛋白的病毒载体有可能通过BMP信号通路促进成牙本质细胞的功能,形成第三期牙本质以保护牙髓。针对第三期牙本质在牙体牙髓病发生、发展和治疗中的作用,根据VEGF、BMP2在牙髓干细胞和成牙本质细胞增殖分化中的作用,在腺病毒介导VEGF和BMP2促成骨作用研究的基础上,提出了我们的研究,即应用腺病毒介导VEGF和BMP2基因转染牙髓干细胞,并检测对牙髓干细胞向成牙本质细胞分化的影响,并且将腺病毒介导的VEGF和BMP2应用于盖髓的动物实验,研究其对第三期牙本质形成的影响,这将为临床上应用VEGF和BMP2治疗深龋和意外穿髓奠定基础。首先,我们从人脱落乳牙牙髓中采用组织块法经酶消化提取、分离和培养牙髓细胞(人乳牙h DPCs)。所有实验均使用第3代牙髓细胞。在成脂诱导培养基(含IBMX0.5mmol/L、地塞米松1μmol/L、胰岛素10mg/L、消炎痛200μmol/L)内培养35天,油红O染色阳性。在矿化诱导培养基中,茜素红染色阳性。表示所培养的牙髓细胞中含有干细胞,并有一定的分化能力。第二,将携增强型绿色荧光蛋白的腺病毒载体(Ad CMV-EGFP)以不同的病毒数量(0、400、800、1000 particles/cell)转导第3代人乳牙h DPCs。在荧光倒置显微镜下观察细胞在转导后的24小时就有EGFP的表达,Ad CMV-EGFP的转导效率随病毒数量的增加而逐渐增加。在病毒量为1000 particles/cell时,细胞增殖没有明显受到影响,细胞的形态无明显变化,病毒转导3d后,转导效率可达70%以上。在以后的实验中,转染Ad CMV-h VEGF或Ad CMV-h BMP2时也选择该浓度作为转导条件进行转导。Ad CMV-h VEGF或Ad CMV-h BMP2转导后3天的细胞提取总RNA,经RT-PCR扩增、2%琼脂糖凝胶电泳,观察到h VEGF和h BMP2表达增加,表明Ad CMV-h VEGF或Ad CMV-h BMP2已成功转导入细胞内并且有m RNA水平的转录。第三,按照病毒数量为1000particles/cell转导,将Ad CMV-h VEGF或Ad CMV-h BMP2对人乳牙h DPCs的作用分别与Ad CMV-EGFP组进行了一系列细胞分子学方面的比较。与Ad CMV-EGFP对照组相比,实验组Ad CMV-h VEGF或Ad CMV-h BMP2转导后,在矿化诱导培养基(含抗坏血酸50mg/L、β-甘油磷酸钠10mmol/L、地塞米松10-8mol/L)中,培养14天和28天进行茜素红染色,有更多的矿化结节形成。矿化诱导7天、14天和21天均检测到细胞碱性磷酸酶活性增高,有统计学意义(P<0.05)。矿化诱导3天、7天和14天提取细胞总RNA,进行RT-QPCR,结果显示,参与骨生成/牙本质发生相关的基因表达大幅度增加,有统计学意义(P<0.05)。Ad CMV-h VEGF在7天时差异更为显著,Ad CMV-h BMP2则在14天时更显著。第四,在体内,我们制备了第三期牙本质修复的动物模型。将Ad CMV-h VEGF或Ad CMV-h BMP2置于大鼠磨牙窝洞内,采用组织学和免疫组织化学观察h VEGF和h BMP2应用对第三期性牙本质形成的作用。体内实验结果同样表明,它们可促进成牙本质细胞增殖和新血管形成,并极大地提高了牙髓中第三期牙本质的形成。本研究采用腺病毒载体介导h VEGF或h BMP2基因转导人乳牙h DPCs,研究了腺病毒载体基因转导h DPCs的可行性,以及h VEGF或h BMP2转导对h DPCs功能的影响。我们的体外和体内的数据表明,Ad CMV-h VEGF或Ad CMV-h BMP2可促进第三期牙本质的形成,具有潜在的临床应用价值。本研究的创新点在于将腺病毒为载体的h VEGF或h BMP2转导用于第三期牙本质形成和修复的研究。更多还原

【Abstract】 Up-to-date dentists still face a challenge saving patients’ pulp without root canal therapy when treating pulp disease.In the tooth, there are four major components: enamel, cementum, dentin, and pulp. The rigid dentin provides powerful protection for dental pulp and support for enamel. Caries, trauma, abrasion and other damage can cause tooth hard tissue defects and pulpitis. One of the main reaction of odontoblasts on dental pulp damage is to form tertiary dentin and then to protect the pulp from further damage. Therefore, how to promote the formation of tertiary dentin and save the vitality of pulp is an important scientific problems.The tertiary dentin formation include the migration of dental pulp stem cell, the proliferation and differentiation of the odontoblasts, and the secretion of dentin matrix. The differentiation of dental pulp stem cells to the odontoblasts is an important process in reparative dentine formation. A variety of factors, cell factors and signaling pathways involve in and control the process. Growth factors can promote the formation of tertiary dentin by promoting the proliferation, differentiation and function of odontoblasts. Studies have showed that VEGF can increase the Runx2 protein by preventing Runx2 protein degradation contributing to Dspp expression.VEGF, the best-characterized angiogenic factor, with endothelial cell specific mitogen activity,can promote the angiogenesis and increase the permeability of vessels in vivo. Therefore, the application of VEGF may contribute to the tertiary dentin formation by promoting the proliferation and differentiation of odontoblasts and then protect the pulp. Bone morphogenetic protein 2(BMP2), belongs to the TGF-β superfamily of proteins. BMP2 is an autocrine secretory protein. BMP2 can promote odontoblastic differentiation and reparative dentin formation through the Smad 1/5, p38 a MAPK and other signaling pathways. Odontoblasts can produce and secrete BMP2 which directly affects the secreting and the adjacent odontoblasts to stimulate proliferation and differentiation of odontoblasts, resulting in dentin formation. Studies have been done to form the tertiary dentin by promoting BMP2 protein expression or applying exogenous BMP2 protein.But BMP2 protein is expensive. The application of viral vector carrying BMP2 gene may promote the functions of odontoblasts by BMP signaling pathways and form the tertiary dentin to repair the pulp damage.For the role of tertiary dentin in pulp disease treatment, according to the effects of VEGF and BMP2 on dental pulp stem cells and the proliferation and differentiation of odontoblasts, based on the osteogenesis study of adenovirus mediated VEGF and BMP2, our research was proposed. This will build the foundation treatment of tooth pulp disease for clinical application of VEGF and BMP2.First of all, we isolated and cultured dental pulp cells from normal human exfoliated deciduous incisors(h DPCs). The third generations of dental pulp cells were used in all experiments. The h DPCs were cultured for 5 weeks in an adipogenic induction media containing 0.5 m M isobutylmethylxanthine, 1 μM dexamethasone, 10 mg/L insulin, and 200 μM indomethacin and then stained with Oil Red O. The positive lipid droplet could be observed. In the mineralized induced medium, the cells also could be observed positive by alizarin red staining. This showed the cultured dental pulp cells contained stem cells and had ability of differentiation.The third passage cells were transduced with recombinant adenovirus vector encoded with enhanced green fluorescent protein(EGFP), Ad CMV-EGFP, at 0, 400, 800 or 1000 particles/cell. The transduced efficiency of adenoviral vector was increased from 0 particle/cell to 1000 particles/cell without affecting the proliferation of h DPCs. Considering the toxicity of adenovirus, the h DPCs were transduced with Ad CMV-EGFP at 1000 particles/cell. About 70% of h DPCs were EGFP-positive on day 3. Finally, VEGF and BMP2 expression was increased after the h DPCs were transduced with Ad CMV-h VEGF at 1000 particles/cell. This indicates that adenoviral vector can be used to transduce h VEGF and h BMP2 into these cells.After transduced with adenoviral vector at 1000 particles/cell and cultured in the mineralized induced medium(containing 10 m M/L sodium β-glycerol phosphate, 50 mg/L L-ascorbic acid, and 10-8 M/L dexamethasone), the comparison of the effects of h VEGF or h BMP2 on h DPCs with EGFP was conducted in a series of cellular and molecular aspects,respectively. Data demonstrated that calcium deposition occurred in the h DPCs, whereas the Ad CMV-h VEGF or Ad CMV-h BMP2 treated group had significantly more Alizarin red S positive cells or mineralized nodule on days 14 and 28 compared to the Ad CMV-EGFP control group. The experimental group also increased ALP activity after 7 days post-culture(P<0.05). RT-QPCR assays show that h VEGF or h BMP2 significantly increased the gene expressions of osteogenic/odontogenic gene markers compared to Ad CMV-EGFP group(P<0.05). The Ad CMV-h VEGF group increased on days 7 is more significant, while Ad CMV-h BMP2 is more significant on days 14.We prepared a rat model to evaluate the tertiary dentin in vivo. The formation of tertiary dentin was observed using histological and immunohistochemical methods after Ad CMV-h VEGF or Ad CMV-h BMP2 adenovirus carrier applied on the surface of prepared cavities. Data from in vivo assays indicated that h VEGF and h BMP2 enhanced pulp cell proliferation and neovascularization,and dramatically increased formation of tertiary dentin in dental pulp.The innovation of this research is to study the effects of h VEGF and h BMP2 transduction mediated by adenovirus vector on the formation of tertiary dentin and the dentinogenic differentiation of h DPCs. We study the feasibility of gene transduction into h DPCs with adenovirus vector and the influence of h VEGF or h BMP2 transduction on the function of h DPCs. Our in vitro and in vivo data suggest that Ad CMV-h VEGF and Ad CMV-h BMP2 have potential clinical application and provides solid evidence to allow us to move to the next level in treating dental pulpitis.更多还原