歯髄幹細胞および歯髄組織のCD146 mRNA発現に対するlipopolysaccharide刺激の影響  [in Japanese] Effects of Lipopolysaccharide-stimulation on CD146 mRNA Expression in Dental Pulp Stem Cells and Dental Pulp Tissues  [in Japanese]

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Author(s)

    • 末山 有希子 Yukiko SUEYAMA
    • 新潟大学大学院医歯学総合研究科 口腔健康科学講座 う蝕学分野 Division of Cariology, Operative Dentistry and Endodontics, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences
    • 金子 友厚 Tomoatsu KANEKO
    • 新潟大学大学院医歯学総合研究科 口腔健康科学講座 う蝕学分野 Division of Cariology, Operative Dentistry and Endodontics, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences
    • 伊藤 崇史 Takafumi ITO
    • 新潟大学大学院医歯学総合研究科 口腔健康科学講座 う蝕学分野 Division of Cariology, Operative Dentistry and Endodontics, Department of Oral Health Science, Niigata University Graduate School of Medical and Dental Sciences
    • 興地 隆史 Takashi OKIJI
    • 東京医科歯科大学大学院医歯学総合研究科 口腔機能再構築学講座 歯髄生物学分野 Department of Pulp Biology and Endodontics, Division of Oral Health Sciences, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University

Abstract

 目的 : 炎症性刺激が幹細胞の動態や遺伝子発現に及ぼす影響については, 依然として不明な点が多い. そこで本研究では, 幹細胞分化能マーカーの一つとして知られるCD146に着目し, ヒト脱落乳歯歯髄幹細胞およびラット骨髄間葉系幹細胞のCD146 mRNA発現に対するlipopolysaccharide (LPS) 刺激の影響を<i>in vitro</i>で検討した. さらに, LPSにより歯髄炎を誘発したラット切歯歯髄組織を検索対象として, <i>in vivo</i>におけるCD146 mRNA発現レベルを併せて解析した. <br> 材料と方法 : ヒト脱落乳歯歯髄幹細胞およびラット骨髄間葉系幹細胞を播種後, LPS (最終濃度 : 10ng/m<i>l</i>) もしくはコントロールとして生理食塩液を培養液に滴下し, 3, 12時間あるいは24時間培養した. 所定時間経過後, 各培養幹細胞から全RNAを抽出したのち, CD146 mRNAの発現をreverse transcription polymerase chain reaction (RT-PCR) 法により半定量的に解析した. また, Wistar系雄性ラットに全身麻酔を施した後, 下顎切歯歯冠を水平方向に切断し, Kファイルにて歯髄腔内に5mm穿掘形成したのち, LPS (1mg/m<i>l</i>) を1μ<i>l</i>貼付し, 窩洞を封鎖した. LPS刺激3, 12, 24時間経過後に切歯歯髄を摘出し, 前述と同様に全RNAを抽出後, RT-PCRを用いてCD146 mRNAの発現解析を実施した. <br> 成績 : ヒト脱落乳歯歯髄幹細胞において, LPS刺激12, 24時間経過後にCD146 mRNA発現の有意な亢進が観察された. また, ラット骨髄間葉系幹細胞ではLPS刺激24時間経過後にCD146 mRNA発現の有意な亢進が観察された. さらにラット切歯歯髄組織においては, LPS刺激3時間経過後にCD146 mRNA発現の有意な亢進が観察された. <br> 結論 : LPS刺激により, ヒト脱落乳歯歯髄幹細胞, ラット骨髄間葉系幹細胞およびラット切歯歯髄組織にCD146 mRNAの有意な発現亢進が認められた.

 Purpose: It is still unclear how inflammatory stimuli influence the kinetics and gene-expression profiles of stem cells. In this study, we focused on CD146, a marker of stem cell differentiation, and aimed to analyze the effect of lipopolysaccharide (LPS) stimulation on CD146 mRNA expression in stem cells from human exfoliated deciduous teeth (SHED) and rat bone marrow mesenchymal stem cells (RBMMSC) <i>in vitro</i>. We also analyzed the level of CD146 mRNA expression in LPS-stimulated rat incisor pulp tissues <i>in vivo</i>.<br> Materials and Methods: SHED and RBMMSC were seeded, and cultured for 3, 12, and 24 hours in the presence of LPS (final concentration 10 ng/m<i>l</i>) or saline (control). After the given period, total RNA was extracted from the samples and CD146 expression was analyzed semi-quantitatively by reverse transcription polymerase chain reaction (RT-PCR). For <i>in vivo</i> analysis, male Wistar rats were anesthetized, the crowns of their lower incisors were cut off horizontally, and the pulp chamber was enlarged at 5 mm in length with K-files. Then, 1 μ<i>l</i> of LPS (1 mg/m<i>l</i>) was applied and the cavities were sealed. At 3, 12, and 24 hours after the LPS stimulation, the pulp tissues were retrieved, total RNA was extracted, and RT-PCR analysis was performed in the same way as mentioned above.<br> Results: After the LPS stimulation, CD146 mRNA expression was significantly upregulated in SHED at 12 and 24h, and in RBMMSC at 24h. In the rat incisor pulp tissue, CD146 mRNA expression was significantly upregulated at 3h after LPS stimulation.<br> Conclusion: LPS stimulation significantly upregulated the expression of CD146 mRNA in SHED, RBMMSC, and rat incisor pulp tissues.

Journal

  • The Japanese Journal of Conservative Dentistry

    The Japanese Journal of Conservative Dentistry 58(4), 282-289, 2015

    The Japanese Society of Conservative Dentistry

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