ABSTRACT
Objective:To develop a simple culture and purifying method for rat dental follicle cells.Methods:The upper and lower first and second intact molar germs of SD rat were separated. Then, dental follicle and enamel organ were stripped together, minced into little pieces, digested with collagenase and cultured. Dental follicular cells were purified by differential passage and indentified by immunohistochemical staining of vimentin and cytokeratin. Results:The primary cells were mixed, consisting of dental follicle cells and enamel organ cells. After differential passage, the cells of fourth passage became purified dental follicle cells. Purified dental follicle cells were elongated spindle or triangle in shape, positive for vimentin and negative for cytokeratin.Conclusion:Dental follicle cells can be purified by several differential passages from the mixed primarily cultured cells.
ABSTRACT
Objective:To study the multiple alternative sp l iceosome of nfic gene in postnatal rat molar tissues. Methods: 3 d postnatal SD rat molar tissues were removed and mRNA was obtained by magne tic beads coupled with oligo-dT18. Two pairs of primers specific to the nfic gene were designed, and nfic gene in the rat molar tissue was amplified b y RT-PCR method.The fragments were inserted into competent DH5? bacteria.Posit ive clones were selected randomly and evaluated by enzyme digestion and sequenci ng.Results:Three alternative spliceosomes of nfic gene with the size of 1.5 kb,900 bp and 650 bp respectively were obtained. The spliceosome s were named rNFIC-1、 rNFIC-2 and rNFIC-3 respectively.Conclusion s:The nfic gene is expressed in postnatal rat molar, and there is a multiple alternative splicing way for the nfic gene to play function.
ABSTRACT
Objective:To investigate the temporal and spatial distrib ut ions of collagenⅠ and Ⅲ during mouse tooth germ development and their function s during tooth mineralization.Methods:Immunohistochemistry stain ing technique was used to test the expressions of collagen Ⅰ and Ⅲ during mous e tooth germ development. Results:collagen Ⅲ was positive in or al epithelial cells in bud stage,in oral epithelial cells and in stellate reticu lum cells in cap stage. During bell and differentiation stage,collagen Ⅲ was positive in oral epithelial cells, stellate reticulum cells, dental papilla cell s and dental sac cells. During P2-10 d(crown development stage), collagen Ⅲ was expressed possitively in ameloblasts,enamel matrix,odontoblasts,predentin, dental papilla cells,dental sac cells and pulp tissues. During P10-30 d(toot h root development stage),collagen Ⅲ was strongly positive in Hertwig's epithe lial root sheath, cementum, alveolar bone and periodontal ligament cells apart f rom above mentioned cell types. CollagenⅠ was not expressed in bud stage and wa s positive in oral epithelial cells,stellate reticulum cells in cap stage. Durin g bell and differentiation stage,collagen Ⅰ was positive in oral epithelial ce lls, stellate reticulum cells, dental papilla cells and dental sac cells.After P2 d (crown and root development stage), the distribution and expression of co llagen Ⅰ were similar to those of collagen Ⅲ.Conclusions:Coll agenⅠand Ⅲ are involved in tooth germ and tooth tissue development. But the fu nction of collagenⅢ is more extensive than that of collagenⅠ.
ABSTRACT
Objective:To detect the expression of upstream stimulatory factor 1(USF1) and its tempo-spatial distribution during mice teeth development. Methods: Total protein was extracted from P1 and P11 d mice first molar teeth germ, and Western blot for USF1 was undertaken. Paraffin sections of first molar teeth germs from E13, 16, 19, P1, 5, 8, 11, 21 d and 6-month-old adult mice were prepared respectively and immunohistochemical staining was carried out. Results: Western blot analysis identified one Mr 43 000 protein from P11 d mice teeth germs, but none from P1d mice. Immunohistochemically, evidently positive staining for USF1 in mice teeth germs began from P5 d, and extended to P11 d, which was mainly confined to the cytoplasm of secreting ameloblasts and odontoblasts, but no staining in bud, cap and early bell stages of tooth germ. However, after tooth eruption on P21 d, USF1 became negative again, although it was still positive in the adjacent muscles, and the same result was observed in adult mice tooth. Conclusion: USF1 is expressed in tooth germ, which localizes solely in secreting ameloblasts and odontoblasts, and its expression was quite dynamic during mice tooth development.
ABSTRACT
Objective:To evaluate the physiological roles of bmp3,bmp4 and bmp7 during the formation of permanent tooth roots in dog. Methods:The expression of bmp3,bmp4 and bmp7 mRNA at different stages of the development of permanent tooth roots was examined by in situ hybridization in 3 dogs aged 12-18 weeks. Results:bmp3 was found in dental sac surrounding the germs at the early stage of tooth root development, and in cementoblasts and periodontal cells at the later stage. bmp4 was found in odontoblasts, dental papilla and osteoblasts. bmp7 positive signals was found only in epithelial cells of root sheath around cervical circulus at early stage, then located in cementoblasts and odontoblasts at later stage. Conclusion:The spatiotemporal expressions of bmp3,bmp4 and bmp7 are widely diverse, indicating that they participate in the regulation of tooth root development.
ABSTRACT
Objective:To investigate spatiotemporal expression of ADAM28 in mouse tooth germ development.Methods:Immunohistochemistry and image analysis technique were used to observe the expressions of ADAM28 at mouse tooth germ development stages.Results:Different expression levels of ADAM28 at tooth germ development stages were observed.At cap stage,ADAM28 was found strongly positive in oral epithelial,stellate reticulum cells of enamel organ,basement membrane,dental papilla cells and dental sac cells.At late bell stage,positive staining was found in ameloblasts,enamel matrix,epithelial root sheath and dental papilla cells.At crown and root development stage,positive staining for ADAM28 was detected in ameloblasts,odontoblasts,cementoblasts,epithelial root sheath,dental papilla cells and dental sac cells.Conclusion:ADAM28 participates in crown and root morphogenesis process ranging from bud stage to late bell stage and from matrix secretion to sclerous tissue formation.It might play an important role in early formation,proliferation and differentiation of odontogenic mesenchymal cells.
ABSTRACT
Objective:To prepare and identify a polyclonal antibody againstadam28 gene product.Methods:Theprotein coding region of ADAM28 was amplified by RT-PCR and cloned into pMD18-T Vector to produce the newconstruct, pMD18-T-adam28. The cloned ADAM28 segment was cut with two restriction enzymes and theadam28fregment was directed into the prokaryotic expression vector, pGEX-4T-1,to produce the expression vector pGEX-4T-adam28. The recombinant plasmid was transformed intoE. coliDH5?and GST-ADAM28 fusion protein was ob-tained after the inducement by IPTG. The fusion protein was extracted and purified by SDS-PAGE,and the newpro-tein band of 35 300 was isolated as antigen, the antigen was injected into rabbits to produce polyclonal antibody a-gainst ADAM28 product.Results:The expression vector pGEX-4T-adam28 was constructed successfully,and GST-ADAM28 fusion protein was obtained. The rabbit serum containing polyclonal antibody against ADAM28 productwas obtained and the antibody was purified by salting out method. Western blot analysis displayed that the antibodyhad high specificity. ELISA analysis confirmed that the titer for the antibody reached 1∶16 000.Conclusion:Thepolyclonal antibody against ADAM28 product with high titer is successfully prepared,it may be used for further studyof the role and expression of ADAM28 during tooth development.
ABSTRACT
Objective:To observe the dynamic process of the development of dentin and enamel during their mineralization period. Methods: Dental germs in maxillae and mandibulae were obtained from spontaneously aborted 21 weeks and 32 weeks fetus. The samples were prepared and stained with modified Mallory's trichrome staining and observed under light microscope. Results: Different mineralized layers in different period of development appeared different color variation in the dental germs. Conciusion: Modified Mallory's trichrome staining method may be used to study the mineralization of dental germ.