レーザーキャプチャー顕微鏡を用いたマウス・セメント芽細胞および歯根膜細胞における遺伝子発現の比較検討

Abstract

Cementum is an essential tissue to maintain tooth function and should be closely correlated to tooth root development and periodontal tissue regeneration. However, detailed features of the periodontium including cementum and specific markers for cementoblasts are unknown. Moreover, the molecular mechanism of periodontal tissue development, homeostasis and regeneration remains unknown. Previous studies have usually examined cementum or periodontalligament (PDL) tissue obtained by manual curettage, resulting in difficulties in isolating pure cementum or PDL. We employed laser capture microdissection (LCM) to isolate cementoblasts and PDL cells from undecalcified frozen sections of murine mandible and to obtain RNA of good quality for subsequent genetic analysis. Over 500 cementoblasts and PDL cells were separately laser captured under microscopy. A bioanalyzer detected peaks of 18S and 28S rRNA both in the laser-dissected cementoblasts and in PDL cells, suggesting that the RNA was of sufficient quality. The RNA samples were amplified due to their small amount and a comparative analysis of mRNA expression by GeneChip showed that about 2,000 genes were differentially expressed between cementoblasts and PDL cells. Both cementoblast-positive and PDL cell-negative genes were serially analyzed by quantitative RT-PCR using RNA samples obtained from mandibles and femurs. Several genes were expressed at higher levels in the mandible than in the femur, suggesting that some might be cementoblast-specific markers. We established a novel experimental system with which to isolate target tissues from single cells in undecalcified frozen sections and to obtain intact RNA. These methodologies could be useful for further investigation of mineralized tissues and to explore tissue-specific factors.