Effects of glypican-1 gene on the cultured rat pulp cells

We prepared the cDNA library as odontoblast-like cell enriched cDNA in vivo with mRNA from the laser irradiated pulp of a rat incisor. The glypican-1 (gpc-1) gene was detected among the identical genes in this cDNA library. GPC-1 is a member of a family of glycosylphosphatidylinositol anchored cell surface heparin sulfate proteoglycans.

Objectives: The effects of glypican-1 gene on the odontoblast-like cell differentiation and the formation of reparative dentin in the rat pulp cells were investigated using down-regulation of gpc-1 gene expression by its specific siRNA.

Methods: The mandibles were removed from 3 male wister rats (5 weeks old) from the same litter. The pulp tissues were cut into several pieces and incubated in a sterile enzyme solution containing 0.1 % collagenase, 0.05 % trypsin. The six different cell populations released were cultured in a culture dish containing a-MEM, 10% heat-inactivated CS, and antibiotics (100£ mg/ml penicillin G, 100 IU/ml streptomycin). After having reached a subconfluent state, the cells were removed from dish and inoculated into 12-well plates for si RNA transfection in a density of 10⁴ cells/cm².

After 24 h the culture medium was replaced with mineralizing medium (10% heat-inactivated CS, 300 £mg/ml glycerophosphate, 50 £mg/ml ascorbic acid, and antibiotics). Pulp cells in a well of 12-well plate were transfected with 1.5 mg of designated pBAsi plasmids using TransIT®-LT1 transfection reagent (Mirus Bio. Co. Ltd, WI, USA). Real-time PCR assays were performed with the primer pairs for gpc-1, tgf-b1, dspp and gapdh.

Results: Down-regulation of gpc-1 gene expression by its specific siRNA in the pulp cells resulted in increased tgfb-1 expression and decreased dspp expression.

Conclusion: These findings suggest that gpc-1 gene might be associated with differentiation of odontoblast-like cells and formation of reparative dentin.