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The Effect of LRAP on Enamel Organ Epithelial Cell Differentiation

Department of Orofacial Sciences, University of California at San Francisco, 513 Parnassus Avenue, P.O. Box #0422; San Francisco, CA 94143–0422, USA

View larger version (17K): [in this window] [in a new window]   Figure 1. The effect of rH58 on enamel organ epithelial cell proliferation. Exogenous rH58 at various concentrations added to enamel organ epithelial cell cultures had no significant effect on cell proliferation, as measured by an ELISA BrdU-labeling cell proliferation assay (p > 0.05, N = 3). The average RFU ± SD values obtained for cell treatments of rH58 at 0 nM, 30 nM, 90 nM, and 180 nM were 162.8 ± 9.4, 194.3 ± 18.1, 214.4 ± 61.4, and 216.6 ± 12.8, respectively.

View larger version (82K): [in this window] [in a new window]   Figure 2. Detection of LAMP-1 in the stratum intermedium of human enamel organ. (A) Immunofluorescent staining of LAMP-1 showed a strong positive staining for LAMP-1 in the stratum intermedium (SI) and stellate reticulum (SR) of a frozen section of a human enamel organ, while immunostaining for LAMP-1 in ameloblasts (Am) was not detectable. (B) Immunostaining of the human enamel organ showed that Notch1 was weakly positive in the SR, while it was hardly detectable in the SI and differentiated Am. (C) The positive control showed strong immunostaining for cytokeratin-14 in the SI and SR, but weaker staining in the Am. (D) The negative control stained with the pre-immune IgG was immunofluorescent-negative. Cell nuclei were counterstained with Hoechst dye (blue).

View larger version (20K): [in this window] [in a new window]   Figure 3. Expression and immunostaining of LAMP-1 in human enamel organ epithelial cell cultures. (A) Reverse-transcription (RT)-PCR product showed positive LAMP-1 mRNA expression in human enamel organ epithelial (EOE) cells, indicated by a 513-bp band in 1% gel. Human enamel organ cDNA library was used as a positive (+) control, while 2 µL of sterile dH2O was used as a negative (–) control. (B) Immunofluorescence of enamel epithelial cells in vitro showed a weak positive staining for LAMP-1. (C) Pre-immune IgG staining of enamel epithelial cells showed immunonegativity for LAMP-1.

View larger version (57K): [in this window] [in a new window]   Figure 4. Characterization of rH58-treated enamel organ epithelial cells. Phase-contrast images showed that, after treatment with rH58 (A), enamel organ epithelial cells became rounded and larger, while the untreated control cells (B) retained their small, cobblestone-like shapes. Immunofluorescent results indicated strong positive staining for LAMP-1 (C) and amelogenin (E), but decreased immunostaining for Notch1 (G) in rH58-treated epithelial cells. The untreated control cells were stained weakly for LAMP-1 (D) and were immuno-negative for amelogenin (F), but were immunopositive for Notch1 (H). Cell nuclei were counterstained with Hoechst dye (blue). Bars = 50 µM.

Journal of Dental Research, Vol. 86, No. 11, 1095-1099 (2007)
DOI: 10.1177/154405910708601114


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