【作者】 杨帆；

【导师】 肖明振；

【作者基本信息】 中国人民解放军第四军医大学 ， 口腔临床医学， 2003， 博士

【摘要】 FGF18是一个新近克隆的FGF家族成员，1998年首次应用PCR方法从鼠胚(E14.5)中分离出来，与FGF8和FGF17属于同一亚家族。研究证实FGF18是一种发育组织重要的分泌性信号分子，在骨骼和软骨的发育中发挥着重要的作用，单纯FGF18的缺失就会严重影响成骨和软骨的形成，那么作为同样具有矿化能力的牙齿，FGF18是否也参与调控其发育和损伤修复的过程呢?本研究带着这个疑问对FGF18在牙胚发育和牙髓组织中的作用进行探索性研究，为今后的研究奠定理论基础和实验依据，具体内容包括： 一、小鼠牙胚FGF18的基因克隆和序列分析 采用异硫氰酸胍一步法从新生小鼠牙胚组织中抽提总RNA，用Oligo(dt)作引物逆转录合成牙胚cDNA，然后利用巢式PCR技术，根据已公布的FGF18序列，设计一对引物从cDNA中扩增出约621bp的特异片段，将扩增的基因片段插入pGEM-T克隆载体，转化大肠杆菌DH5α，挑选阳性克隆，提取质粒DNA，通过限制性酶切进行鉴定并进行核苷酸序列测定。结果表明：成功克隆到小鼠牙胚FGF18全编码区基因片段，序列与GenBank中收录的FGF18cDNA序列一致，初步说明FGF18参与牙胚的发育。 二、FGF18在牙胚发育和牙髓组织中的作用 首先设计一对去除FGF18信号肽基因序列的引物，然后利用PCR技术从 第四军医大学博士学位论文小鼠牙肛 CDNA中克隆约 524hp的编码区片段，并连人表达载体 pRSETB中，转化大肠杆菌BLZI（DE3）plySS＊ IPTG诱导表达后，12％SDS－PAGE凝胶电泳鉴定，发现在相对分子量为 22KD处出现预计的 FGF融合蛋白表达条带，经大量诱导表达和 Ni－NTA柱亲和层析纯化，获得了高纯度的 FGF原核表达产物。以 FGF原核表达产物为抗原，混入完全弗氏佐剂，免疫新西兰大白兔，将获得的多克隆抗血清经硫酸胺初步纯化后，ELISA实验检测抗体效价，Western Blot检测抗体的特异性，结果表明成功制备兔抗鼠 FGF多克隆抗体。 然后采用自行制备的抗体对牙胚发育和牙髓组织中 FGF的时空表达形式进行免疫组化研究。 l、在牙胚发育中：蕾状期FGF18在成釉器和口腔粘膜上皮细胞表达阳性，而在周围密集的间充质细胞表达阴性；帽状期在内釉上皮、外釉上皮、牙胚间充质和牙囊未见阳性表达；钟状早期牙胚，FGF在内釉上皮细胞和外釉上皮细胞阳性表达，牙乳头细胞弱阳性表达，星网状层和中间层细胞阴性，牙囊细胞表达阳性，在牙槽骨和软骨中呈阳性表达；钟状晚期，内釉上皮和外釉上皮分化为成釉细胞和成牙本质细胞，并分泌釉质和牙本质基质，此时 FGF在具有分泌功能的成牙本质细胞和成釉细胞表达强阳性，牙乳头细胞和牙囊细胞阳性，星网状层阴性。提示FGF18可能参与牙胚的发生、细胞分化和基质矿化，但对牙胚的形态发生可能作用较小。 2、在牙髓组织中：正常牙髓成牙本质细胞、牙髓细胞以及血管内皮细胞中未见有 FGF阳性表达，但在炎症组 FGF在成牙本质细胞和血管内皮细胞阳性表达，牙髓细胞弱阳性，表明 FGF可能参与牙髓炎症和损伤修复三、FGF18对牙乳头细胞作用机制的研究 首先设计一对引物，用于构建 FGFIS真核表达载体，利用 PCR技术从新生小鼠CDNA中克隆540hp的FGF18基因片段，然后将片段连入真核表达载体psecTagZB 中，构建psecTagZB－FGF18重组质粒，在LipofectAMINEm 2000 4 第四军医大学博士学位论文脂质体介导下将psecTagZBF 重组质粒转染进哺乳动物细胞COS刁，通过ELISA方法鉴定COS－7上清中的FGF18真核表达产物。采用MTT法、酶动力法以及免疫组化方法检测COS－7上清中FGF18对牙乳头细胞增殖、ALPase以及 1型胶原合成的影响，结果发现 FGF显著促进人牙乳头间充质细胞的增殖能力（P＜0刀5），并增加 ALPase的分泌（P＜0刀5，这种作用呈一定的时间、剂量依赖性，此外F＊F18可明显增加细胞1型胶原的合成（P＜0刀1），提示 FGF在人牙乳头间充质细胞的分化和矿化过程中可能起着重要的调节作用，但它的确切调控机制仍需一些更能模拟体内牙乳头细胞分化过程的实验加以验证。 综上所述，FGF参与了牙胚的发育，并在牙胚的发生、成牙本质细胞分化和基质矿化中发挥着一定的调控作用，此外 FGF还参与牙髓炎症和损伤修复。今后的工作应针对其调控作用的分子机制进行更深入的研究，以全面揭示 FGFIS的功能作用。更多还原

【Abstract】 Fibroblast growth factor 18(FGF18) was recently found as a member of FGF family,belonging to the subfamily of FGF8 and FGF 17.It was first cloned from mouse embryo (El4.5) by PCR method in 1998 and was proved to be an important signal molecule during development,expecially bone and cartilage development.we know ,tooth is a mineralized organ,shares many similar things with bone and cartilage during development.Thus,the aim of this research was to investigate whether FGF 18 ws involved in tooth development and how.1. Cloning and sequencing of mouse FGF18 from tooth germIn this study, total RNA was extracted from the mouse tooth germ by acid guandinium thiocyanata-phenol-chloroform method, the desired cDNA products were obtained from the total RNA by RT-PCR with the primers including Olido(dt) and two gene specific primers respectively. The segment (about 621bp) was inserted into pGEM-T vector and the recombinant plasmid was transformed into E. coli DH5 a . The positive clones were analyzed with restriction endonuclease mapping and DNA sequencing. The results showed that the nucleotide sequence we cloned were consistent with the GenBank record. These suggested that FGF 18 had effects on tooth development.2. Effects of FGF18 on tooth germ development and dental pulpThe open reading frame of FGF18 cDNA was inserted into prokaryotic expression vector pRSET-B. The recombinant plasmids were then transformed into E. coli BL21 (DE3) plysS and induced by 1M IPTGAfter inductions, new protein bands appeared on SDS-PAGE gel near 22kDa. The expressed product from induced cells was purified and used to immune New Zealand White rabbits together with Freund’s complete adjuvant to produce antibody. The results of ELISA test and Western blot confirmed that we had successfully made FGF18 polyclonal antibody.After that,we studied the expression of FGF18 in mouse developing tooth germ and human healthy or inflamed dental pulp tissues by immunohistochemistry method. (1) Tooth germ development:In bud stage FGF18 was expressed in dental epithelium and enamel organ. No Positive expression was found.in cap stage.In early bell stage, FGF18 was found in both inner/outer enamel epithelial cells and dental sac cells,as well as in osteoblast and chondrocyte,and weak staining in dental papilla cells, negative staining in stratum intermedium and stellate reticulum cells. In late bell stage strong positive expression was found in ameloblast and odontoblasto The results suggested that FGF18 had effects on cell differentiation and matrix mineralization besides tooth initiation (2)Dental pulp:No expression of FGF18 was found in healthy dental pulp, but positive expression was seen in odontoblast and endotheliocyte of blood vessels in inflamed pulp. Weak positive expression was seen in dental pulp cells.These suggested that FGF18 played a part in dental pulp inflammation and reparation after tissues injury. 3. Effects of FGF18 on dental pailla cells in vitroWe constructed eukaryotic expression plasmids by inserting FGF18 cDNA into pSecTag2B vector and transfected the plasmids into COS-7 cells. ELISA testshowed that FGF18 was synthesized and secreted by the cells.Then MTT method and enzyme dynamics method were used to study the effects of FGF18 on dental papilla cells.The results revealed that FGF18 could stimulate dental papilla cells proliferation (PO.05), and increased the alkaline phosphatase (ALP) activity in time and dose-dependent manner(P<0.05). FGF18 could also promoted the expression of type I collagen(P<0.01).These suggested that FGF18 played a role during odontoblast differentiation.更多还原