RNA interference를 이용한 OD314 유전자의 발현억제가 상아질모세포 전구세포에 미치는 영향
Inactivation of the OD314 gene by RNA interference in preodontoblast cell lines
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상아질모세포는 신경능선세포에서 기원하며 상아질의 유기질을 형성하는데 상아질모세포의 분화과정이 나 상아질의 형성과정과 관련한 분자생물학적 기전은 아직 명확히 밝혀져 있지 않다. OD314는 흰쥐의 치아발생 과정과 세포배양실험에서 상아질 형성과정 특히 석회화결절이 형성되는 시기에 그 발현이 현저하며 상아질의 석 회화과정에 중요한 기능을 암시한다. 본 연구에서는 OD314 유전자의 발현을 RNA interference를 이용하여 억제하 여 상아질모세포내 다양한 유전자들의 단백질들을 평가함으로서 상아질의 형성과정에서 OD314의 기능을 알아보 고자 하였다. 1. MDPC23 세포에 ascorbic acid와 β-glycerophosphate 첨가하여 석회화 결절 형성을 유도한 실험에서 배양 후 14 일에 석회화 결절이 관찰되었다. 2. MDPC23 세포의 석회화 결절 형성과정에서 OD314, OC 및 DSPP mRNA는 배양초기부터 석회화 결절이 형성 된 14일에도 동일한 발현을 보인 반면, ON mRNA는 석회화 결절이 형성되기 시작하는 7일부터 발현이 감소되 었다. 3. MDPC23 세포의 배양과정에서 OD314 단백질은 17 kDa 정도의 크기로 배양 시작부터 7일까지 동일한 발현을 보였으며 석회화 결절이 형성된 14일에는 더욱 강한 발현을 보였다. 4. MDPC23 세포에 OD314 siRNA를 transfection한 실험에서 OD314, OC, ON 및 DSPP의 mRNA의 발현이 감소 하였고 OD314의 단백질 발현 또한 OD314 siRNA를 transfection한 세포에서 감소하였다. 이상의 결과를 종합하면 OD314가 상아질모세포의 주요 유전자들의 조절에 관여할 것으로 사료되나 이를 명확히 하기 위하여 앞으로 OD314에 대한 기능과 상아질모세포 관련인자들과의 상호연관성에 대한 향후 보완 연구가 필요할 것으로 사료된다.
Abstract
Tooth development depends on reciprocal interactions between oral epithelium and ectomesenchyme. The ectomesenchyme-derived odontoblasts secrete several collagenous and non-collagenous proteins to form a unique extracellular matrix. OD314 was obtained by subtractive hybridization between odontoblasts and osteoblast/pulp cells, and differentially or predominantly expressed in odontoblasts of rat incisors compared to osteoblasts and pulp cells. However, little is known about the function of OD314 in odontoblast differentiation. In this study, to better understand the function of OD314, we inactivated the OD314 gene in mouse MDPC23 cells using U6 promoter-driven siRNA method. After the characterization of mineralized nodule formation and molecular expression of MDPC23 cell, Inactivation effects of the OD314 were evaluated by RT-PCR and western blot. 1. Mineralized nodule formation was observed after 14 days of culture in MDPC23 cells. 2. The OD314, OC and DSPP mRNA were highly expressed throughout the cultures, while the expression of ON decreased as the MDPC23 cells differentiated. 3. The OD314 protein was weakly expressed at 7 days of culture, but increased gradually as MDPC23 cells reached mineralization stage. 4. The inactivation of OD314 by RNA interference downregulated the expressions of OD314, DSPP, OC, and ON mRNA and OD314 protein in MDPC23 cells. These results suggest that OD314 may play a important role in mineralization process of odontoblast and also regulate odontoblast-related genes such as OC, ON, and DSPP.
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