Complete sealing zone formation needs multiple gene participation

摘要

The mechanism of osteoclasts attachment to the bone surface has not been well understood. In particular sealing zone is a very specific structure of active osteoclast on bone. The molecular basis on its formation has recently attracted much attention in biologists. In this study, we have performed differential display (DD) PCR with the template RNAs extracted from osteoclasts cultured on ivory or collagen type I to identify genes specifically expressed in osteoclasts attached to ivory. Highly enriched osteoclasts were harvested from 10-day-old rabbit according to the method of Kakudo et al. and cultured on each surface. DDPCR was performed using their total RNAs, then PCR products were cloned into pCR-TRAP vector, followed by the DNA sequencing. 22 cDNA fragment clones were found in osteoclast attached to ivory. Among them, 11 clones which located at 3'terminal of related genes were sequenced. Two of them were found as known genes, i.e. PTX3 and DRAK1. We selected 6 clones and examined tissue distribution by slot blot. The data showed that 6 clones expressed in various pattern. These result indicated that different pattern of gene expression was revealed when osteoclast attached to different matrix. It also suggested osteoclasts could form the effective sealing zone with the participation of multiple genes.