【作者】 孙铭；

【导师】 欧阳喈；

【作者基本信息】 吉林大学 ， 口腔临床医学， 2005， 博士

【摘要】 牙周疾病是以牙周支持组织的进行性破坏为特征,最终导致牙齿丧失的一类严重危害口腔健康的疾病。由于牙周疾病的病变涉及的组织范围广,包括牙周韧带、牙骨质和牙槽骨等组织类型,要实现牙周组织的再生性修复一直是临床研究的重点和难点。研究表明,牙周再生是一个十分复杂的、需要多种细胞类型、多种信号系统的参与和调控的过程。而牙周膜细胞是牙周组织再生重要的细胞学基础,是牙周再生研究的核心问题之一。本实验通过体外培养人PDL 细胞,研究PTH 和EMD 在PDL 细胞向成牙骨质/成骨细胞表型转化过程中的调控机制。通过检测细胞增值率、矿化相关蛋白mRNA 的表达量、细胞合成分泌的矿化蛋白含量、碱性磷酸酶酶比活性和体外矿化能力等实验来评价在间歇性PTH、不同浓度的EMD 和两者联合处理PDL 细胞后对细胞表型分化的影响。结果表明,单独间歇性PTH 处理的PDL 细胞在成骨细胞/成牙骨质的表型分化上有重要作用:通过上调矿化蛋白的基因表达,增加相关蛋白的产量来促进PDL 细胞的成骨/成牙骨质能力,其调控方式具有多水平和正负双向性的特点。不同浓度的EMD具有促分化和增殖的双效功能,通过增加功能细胞数量,上调矿化蛋白基因的表达水平和翻译水平来促进PDL 细胞的成骨/成牙骨质表型分化,并在诱导表型转化和调控矿化方式上具有明显的特异性。联合应用实验表明:间歇性PTH 与EMD联合应用对PDL 细胞的成骨/成牙骨质表型分化具有协同效应。提示,一定节律作用的PTH 和适宜浓度的EMD 可以促进PDL 细胞在体外成骨细胞/成牙骨质的表型转化,具有协同效应和双向调节作用的特点。更多还原

【Abstract】 The ideal destination of periodontal therapy is to achieve an anatomicand functional regeneration, which means the regeneration of connectivetissues, the new forming fibrin-bond imbedded in the new depositedaccellular cementum. Actually, the regeneration process are very complexand multilevel regulated one, which includes numerous types cells,systemic hormones and many different unknown protein matrixes, and allof them interacting each other by different ways and different levels, finallyto form a net-like modulate system with the purpose to fulfill the realregeneration. The studies on biological and cytological behaviors of PDLcells show its key role in the process of periodontal regeneration, and alsoacting as a target for various biological stimulators.Recently, the funding of EMD making a peculiar work duringperiodontal restoration improved the concentration of researching on thisfield. It had been demonstrated that EMP could facilitate the PDL cell onthe ability of attachment, stretchment and inducing the PDL cell to secretemany important growth factors and cytokines which can improve themicro-environment conditions from time to time. When enhancingproliferation, EMP can improve the ALPase synthesization and expressionas well. Its biological behavior is of more as multifunctional growth factors.Scholar approved that EMP can induce osteoblastic cell line intoproliferational and differentiational stage. Combined EMP and GTR canstimulate the cementum regeneration at periodontal damnification withnewly forming fibrin embedded. This kind of completely regenerationmimics the structure of original one implies that EMP made a importantpart during this process. Generally speaking, the most important step forperiodontal regeneration is the rebuild of cementum, so the basement of theperiodontal regeneration is to improve the cells can form new cementum.During the development of tooth, the inner cell of Hertwig’s epithelialsheath secrets a kind of matrix protein which belongs to the families ofenamel matrix protein. They may act as a guider for hydroapatite formingand also take part in the periodontal regeneration and cementum forming.Basing on this theory, EMD should be a stimulator to improve theproliferation and differentiation of PDL cells, which can benefits theprocess of new periodontum forming. In vivo studies, EMP can restore thedamaged periontum with histological destinies. Further more, EMD canimpulse relative cells to secrete some important growth factors whichcoordinate in regeneration process. Importantly, the interaction of thesefactors can precisely regulate the behavior of different kinds of cellinvolved in this process. All of these evidences show that EMD make acrucial role in PDL functional behaviors. Parathyroid hormone (PTH), an endocrine factor secreted by theparathyroid glands, is the main hormone regulating bone remodeling.Interestingly, PTH is a double-edged sword for bone metabolism: whereascontinuous infusion of PTH causes bone loss, intermittent administration ofPTH induces bone formation. It is demonstrated that PTH take part in thetooth development in the stage of biomineralization, especially on themature stage, which regulate the interaction between epithelium andmesenchymal. Due to the mature cementum rarely remodeling, the rebuildof it stands out distinctly. The bone forming effect of PTH applies us a newway to periodontal regeneration, especially in cementum. The study here focuses on the phenotype changing duringadministrated PDL cells by PTH, EMD or both. By means of RT-PCR,ElISA, ALP enzyme dynamics and biomineralization in vitro, we try toidentify the possible mechanism on osteoblast and cementoblastdifferentiation. The mineralization relative proteins include ALP, OCN andBSP, to assay the status of PDL cells on the mRNA expression and proteinlevel. The PDL cells harvested from extracted teeth from orthodontic clinicfor therapy reasons, which should be healthy without caries, malformationand periodontisis. The cells were isolated by a sharp scalpel at the middlearea of the root. The tissue was separated into tiny blots with 1mm3. Afterculturing 5-8 days, there are cells grew out of the tissue blots, subcultureand label them. Take the fifth passage cells for trails. Seeding the cells into24-well plates by 6 groups including control and experiment one (n=6). In the trail of intermittently administration PTH for PDL cells, there isno difference on proliferation of PDL among different groups. After 12 daysdealing, ALP mRNA went upper obviously and OCN got the secondposition. It seems no remarkable changes on BSP mRNA. However, ingroup constantly, the mRNA of ALP and BSP were restrained. On proteinassay, the production of OCN increased with dealing time, while increasinghappened in BSP when culture time went ahead. But no changes in controland dealing constantly groups. In enzyme assay trails, the positive lineappeared in groups both 3h and 6 h with no big difference in other groups.About vitro biomineralization trails, only group with 6h dealingintermittently appeared the positive staining area, which characterized asconcentrated and limited areas. In trails of EMD administrated alone, the proliferate effect showedonly in high concentration groups, appearing to be doze-dependedrelationship. The peak point due to 50ug/ml EMD group and 25ug/ml EMDgroup made the second. In mRNA assay, ALP showed a remarkableincrease in every groups whatever in different groups except for controlgroup. The case was similar to mRNA of BSP, however, the total expressionwere nearly 50%-25% less than that of in ALP. Interestingly, there was nodifferent in OCN mRNA in all groups. The accordant situations found inprotein assay. In vitro mineralization trails, the high concentration did work,which shows the positive areas with dispersive and small colons. Combined EMD and PTH, the proliferation were enhanced greatlywith the time going on. The mRNA expression of mineralization proteinshowed the ALP mRNA and BSP mRNA went up with EMD concentrationincreasing, appearing a doze-depended relationship. The climax appearedon 50ug/ml EMD & PTH group. Generally, the expression of BSP mRNAwas much lower than that of ALP. The expression of OCN mRNA got peakon 2.5ug/ml& PTH group with no great changes in other groups. Thefamiliar situation happened in protein assay trails. The vitrobiomineralization trails showed the similar appearance in EMD aloneexperiment: dispersive and small positive colons. A little difference was thenumber and the colon area increasing slightly. Comparing three parts of experiments: on proliferation of PDL cells, theproliferative effect of EMD was enhanced with the existing of PTH whilePTH dealing alone did no difference on proliferastion.On mineralizationalprotein mRNA expression assay, OCN mRNA showed obviouslytime-depended relationship, the peak point arriving at administratedconstantly group. However, the constantly administration on PDL cell更多还原