Cloning And Expression Of Tooth Germ FGF18 And A Pilot Study On Its Biological Effects

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.